Method of suppressing gene transcription through histone lysine methylation

ABSTRACT

The present invention relates to methods of suppressing the transcriptional expression of one or more genes by methylating the chromatin histone proteins of the one or more genes. Specifically, a viral SET domain histone lysine mehtyltransferase (vSET or vSET-like protein) methylates lysine 27 of a gene&#39;s histone protein 3 (H3-K27) thereby suppressing the transcription of the gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of, and claims priority to, U.S.patent application Ser. No. 12/896,510, filed Oct. 1, 2010, which is acontinuation of International Application No. PCT/US2009/039187, filedApr. 1, 2009, which claims the benefit of priority to U.S. ProvisionalApplication No. 61/041,563, filed Apr. 1, 2008, priority to each ofwhich is claimed, and the contents of each of which are herebyincorporated by reference in their entireties.

GRANT INFORMATION

The invention was made with government support under grant numbersGM073207 and CA087658 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods of suppressing thetranscriptional expression of one or more genes by methylating chromatinhistone proteins. Specifically, a viral SET domain histone lysinemethyltransferase (vSET or vSET-like protein) methylates lysine 27 of agene's histone protein 3 (H3-K27), thereby suppressing the transcriptionof a gene or genes.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted herewith via EFS on Jan. 13, 2017. Pursuant to 37 C.F.R. §1.52(e)(5), the Sequence Listing text file, identified asSequence_Listing.txt, is 26,135 bytes and was created on Jan. 13, 2017.The Sequence Listing, electronically filed herewith, does not extendbeyond the scope of the specification and thus does not contain newmatter.

BACKGROUND

Position-specific modifications of histones provide epigenetic controlof gene expression and silencing in eukaryotes (Nightingale et al., CurrOpin Genet Dev 16, 125-36 (2006); Fischle et al., Curr Opin Cell Biol15, 172-83 (2003)). Such modifications include acetylation, methylation,phosphorylation and ubiquitination (Id.). Viruses recruitchromatin-associated transcriptional proteins for their genomemaintenance and replication. For instance, the papillomavirus E2 proteinbinds Brd4 to tether the viral genome to mitotic chromosomes to ensurepersistence of viral episomes in viral infected cells (You et al., Cell117, 349-60 (2004); Wu et al., Genes Dev 20, 2383-96 (2006)). Theadenovirus E1A protein interacts with the retinoblastoma protein p130 todisrupt a network of protein interactions required for silencing ofE2F-responsive genes in quiescent cells (Ghosh et al., Mol Cell 12,255-60 (2003)). Moreover, the trans-activator Tat from humanimmunodeficiency virus recruits the histone acetyltransferases p300/CBP(CREB binding protein) and PCAF (p300/CBP associated factor) for lysineacetylation of a nucleosome, a remodeling step required fortranscriptional activation and replication of the integrated provirus(Mujtaba et al., Mol Cell 9, 575-86 (2002); Don et al., EMBO J 21,2715-23 (2002)). Despite the indirect virus/host protein recruitmentmechanisms to reconfigure chromatin structure for viral transcription,no viral enzymes have been reported that directly modify histones andmodulate host gene transcription. Histone lysine methylation, except forH3-K79, is catalyzed by a family of SET domain proteins first identifiedin Drosophila proteins: Suppressor of variegation, Enhancer of zeste(E(z)) and Trithorax. (Bannister et al., Cell 109, 801-6 (2002); Lachneret al., J Cell Sci 116, 2117-24 (2003)). Position- and state-specifichistone lysine methylation by SET domain proteins in a specificbiological context specifies unique functional consequences (Bannisteret al. and Lachner et al.). For instance, during cell proliferation,H3-K4 di-methylation by Set1 correlates with basal transcription,whereas H3-K4 tri-methylation occurs at fully activated promoters (Simset al., Genes Dev 20, 2779-86 (2006); Wysocka et al., Nature 442, 86-90(2006); Bernstein et al., Proc Natl Acad Sci USA 99, 8695-700 (2002)).Regional H3-K9 tri-methylation by Suv39h at transcriptionally inertchromatin domains is a hallmark of constitutive hetero-chromatin (Peterset al., Nat Genet 30, 77-80 (2002)). During cell differentiation,extended H3-K27 di- and tri-methylation by the Drosophila Polycomb group(PcG) Esc-E(z) complex or the mammalian counterpart Eed-Ezh2 complex arelinked to Hox gene silencing, X-chromosome inactivation, germlinedevelopment and stem cell pluripotency, as well as cancer (Czermin, B.et al. Cell 111, 185-196 (2002); Muller, J. et al., Cell 111, 197-208(2002); Cao et al. Science 298, 1039-1043 (2002); Kuzmichev et al.,Genes Dev 16, 2893-905 (2002); Plath et al. Science 300, 131-5 (2003);Boggs et al., Nat Genet 30, 73-6 (2002); Bernstein et al. Cell 125,315-26 (2006); Boyer et al., Nature 441, 349-53 (2006); Lee et al. Cell125, 301-13 (2006); Cao & Zhang, Curr Opin Genet Dev 14, 155-64 (2004)).

The high degree of modification complexity and coding potential ofhistone lysine methylation in epigenetic control may explain theexistence of an unusually large family of SET domain proteins with morethan 700 members (Schultz et al., Proc. Natl. Acad. Sci. U.S.A. 95,5857-5864 (1998)). Notably, within this extensive family is a smallsubclass of SET domain proteins encoded by viruses and bacteria of whichlittle is known about their cellular functions. One of these viralproteins is the SET domain protein (vSET) encoded by Paramecium bursariachlorella virus 1 (PBCV-1) which specifically methylates Lys27 inhistone 3, a modification implicated in gene silencing (Manzur et al.,Nat Struct Biol., 10:187-196). PBCV-1 is the prototype of a family oflarge, icosahedral, double-stranded DNA—containing viruses that areknown to replicate in certain unicellular, eukaryotic chlorella-likegreen algae, particularly zoochlorellae (Van Etten et al., Annu. Rev.Microbiol. 53, 447-494 (1999)). DNA sequence analysis of PBCV-1 revealsthat this giant virus contains a large 330 kb genome of 376protein-encoding genes (Li et al. Virology 237, 360-377 (1997)). The SETdomain-containing PBCV-1 protein consists of 119 amino acids andrepresents the smallest known SET domain—containing protein in the SETdomain family, although vSET lacks the cysteine-rich pre-SET andpost-SET motifs flanking the conserved core SET domain. These pre-SETand post-SET motifs are required for histone methyltransferase activityin various SET domain proteins including human SUV39H1 (Rea et al.Nature 406, 593-599 (2000)). The presence of SET domain-like proteins inviruses raises questions about whether such proteins have histonemodifying activities, and if so, what are the cellular consequences whenthese proteins are expressed in vivo (Qian & Zhou, Cell Mol Life Sci 63,2755-63 (2006); Manzur et al., FEBS Lett 579, 3859-65 (2005);Alvarez-Venegas et al., Mol Biol Evol 24, 482-97 (2007)).

SUMMARY OF THE INVENTION

The present invention provides a method for selective or generalsuppression or inhibition of gene expression. Specifically, the presentinvention provides a method of suppressing the transcriptionalexpression of a targeted gene in a cell by introducing into the cell aprotein that methylates a chromatin histone protein of the targetedgene.

In non-limiting embodiments, the chromatin histone protein is thehistone 3 protein (H3). In a specific non-limiting embodiment, lysine 27of H3 is methylated (H3-K27).

In non-limiting embodiments, the introduced protein comprises a lysinemethyltransferase.

In particular non-limiting embodiments, the lysine methyltransferase isa histone lysine methyltransferase.

In particular non-limiting embodiments, the lysine methyltransferase isa viral histone lysine methyltransferase.

In preferred specific non-limiting embodiments, the lysinemethyltransferase comprises a Chlorella virus SET domain of a viralhistone lysine methyltransferase protein.

In particular non-limiting embodiments, the present invention provides amethod for suppressing/inhibiting expression of a specific targeted genein a cell by introducing, into the cell, an effective amount of anisolated protein that methylates the targeted gene's H3-K27.

In a further non-limiting embodiment, when a specific gene is targetedfor expression suppression, introduction of the protein into the cellexpressing the specific targeted gene does not suppress the expressionof other genes expressed by the cell.

In one embodiment, H3-K27 is mono-methylated.

In another embodiment, H3-K27 is di-methylated.

In another embodiment, H3-K27 is tri-methylated.

In one embodiment the targeted gene is a cytokine, for example, TNF-α,TGF-β, IFN-γ, IL-2 or IL-10.

In another embodiment, the targeted gene is an oncogenic gene, forexample, MDM2, Src, a Ras kinase, a receptor tyrosine kinase, EFGR,PDGFR or VEGFR. In another embodiment, the targeted gene is ahomeodomain gene, for example, HOXA2, HOXA5, HOXA7, HOXA9, HOXB9, HOXC6,HOXC8, HOXD8, or Hey1.

In another embodiment, the targeted gene is a transcription factor, forexample, myc or NF-κB.

In another embodiment, the targeted gene codes for a receptor, forexample, an Androgen Receptor, Retinoic Acid receptor (RAR), or RetinoicAcid X receptor (RXR).

In another embodiment, the targeted gene is a regulatory gene whichsuppresses the expression of a second gene. Targeting the regulatorygene, according to the present invention, reduces the suppression of thesecond gene, therefore increasing the second gene's expression. In oneembodiment, the second gene is a transmembrane protein which functions,for example, in cell adhesion, or tumor suppression. Such proteinsinclude, for example, E cadherin or M50/Beta-catenin, respectively

In another embodiment, the targeted gene is a regulatory gene whichpromotes the expression of a second gene. Targeting the regulatory geneaccording to the present invention, reduces the expression of the secondgene.

In another embodiment, the targeted gene codes for a protein whichfunction, for example, in regulating cell proliferation. Such proteinsinclude, for example, cyclins such as Cyclin D.

In another embodiment, the targeted gene encodes an HIV transcriptionalactivator protein tat.

In one embodiment, the transcriptional expression of a gene is inhibitedin cells of a multicellular organism.

In another embodiment, the transcriptional expression of a gene isinhibited in a host cell, for example, but not limited to, a cell of aeukaryotic cell line, for example, a breast cancer cell line (e.g. anMCF7 breast cancer cell line or an MCF10A breast cancer cell line), aprostate cancer cell line (e.g. a PC3 prostate cancer cell line or anRWPE prostate cancer cell line), or a leukemic cell line (e.g. a K562leukemic cell line, an HL-60 leukemic cell line, or a U937 leukemic cellline).

In another embodiment, the introduced protein further comprises anuclear localization signal, for example, the amino acid sequenceLys-Arg-Met-Arg (KRMR) (SEQ ID NO:3).

In another non-limiting embodiment, the introduced protein furthercomprises a histone demethylase inhibitor.

In another non-limiting embodiment, the introduced protein is isolatedfrom a chlorella virus, for example, the chlorella viruses PBCV-1,NY-2A, NY-2B or MA-1D.

In another non-limiting embodiment, the introduced protein is a viralSET (vSET) histone lysine methyltransferase domain encoded by a vsetgene isolated from Paramecium bursar Chlorella Virus-1 (PBCV-1).

In another non-limiting embodiment, the introduced protein is avSET-like protein encoded by a vset-like gene isolated from a chlorellavirus.

The invention also provides for expression vectors that encode theintroduced protein (e.g., a recombinant viral histone lysinemethyltransferase, vSET or vSET-like protein), for example, a lentivirus(e.g. lenti-virus vector pLVET-tTRKRAB) or an adenovirus (e.g. adenovirus vector VQpacAd5CMVK-NpA) expression vector, which act as vehiclesfor introduction of the protein into the cell. In further embodiments,the expression vector comprises a cationic lipid, polymer, or liposomecomplex.

The invention also provides for pharmaceutical compositions for humanadministration comprising a purified lysine methyltransferase, histonelysine methyltransferase, viral histone lysine methyltransferase,protein comprising a Chlorella virus SET domain of a viral histonelysine methyltransferase, vSET or vSET-like protein in apharmaceutically acceptable carrier.

The invention also provides for methods of treating a disease ordisorder in which suppression of gene expression can provide atherapeutic benefit. Such diseases or disorders include, but are notlimited to, blood disorders (for example, but not limited to, acutelymphoid leukemia and lymphoma), developmental disorders (for example,but not limited to, mental retardation, epilepsy or movement disorders),inflammatory disorders, cancer (for example, but not limited to, gastriccancer, breast cancer, colorectal cancer, thyroid cancer, ovariancancer, prostate cancer and leukemia), and diseases of the central andperipheral nervous systems wherein over expression of one or more genescontributes to pathology of the disease or disorder. Alternatively, theinvention also provides for methods of treating a disease or disorderwherein reducing the expression of one or more genes can provide atherapeutic benefit even if the disorder is not caused by the overexpression of one or more genes.

The invention also provides methods of treating an individual having adisorder requiring gene therapy, comprising administering to theindividual a composition which comprises a therapeutic vector encoding aprotein of the present application, for example, a protein comprising aChlorella virus SET domain of a viral histone lysine methyltransferase.

In one non-limiting embodiment, expression of the protein carried by thevector results in suppression of a targeted gene or genes whoseexpression contributes to a pathological condition in the individual,for example, but not limited to, cancer. The invention also provides fornucleic acid constructs and expression vectors encoding a protein of thepresent invention (for example, a protein comprising a Chlorella virusSET domain of a viral histone lysine methyltransferase) and a second DNAtargeting protein, wherein the DNA targeting protein enables methylationand transcriptional suppression of a specific target gene.

The invention also provides for fusion proteins a protein of the presentinvention (for example, a protein comprising a Chlorella virus SETdomain of a viral histone lysine methyltransferase) and a DNA targetingprotein, wherein the DNA targeting protein enables methylation andtranscriptional suppression of a specific target gene.

The invention also provides for methods of introducing a protein of thepresent invention (for example, a protein comprising a Chlorella virusSET domain of a viral histone lysine methyltransferase) or an expressionvector that encodes a recombinant protein of the present applicationinto a cell.

The invention also provides for methods of inhibiting the growth cycleof a cell by introducing a protein of the present invention (forexample, a protein comprising a Chlorella virus SET domain of a viralhistone lysine methyltransferase) or an expression vector that encodes arecombinant a protein of the present invention (for example, a proteincomprising a Chlorella virus SET domain of a viral histone lysinemethyltransferase) into a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1e show the presence of vSET in PBCV-1 virions. a, Hosttranscription shutoff by virus infection. Incorporation of ³H-adenineinto RNA in PBCV-1 infected Chlorella NC64A cells as compared tomock-infected cells. b, Detection of vSET in PBCV-1 virions and virusinfected Chlorella cells by immunoprecipitation (IP) and thenimmunoblotting (IB) with anti-vSET antibodies. c, The presence of vSETin PBCV-1 virions is not due to external contamination, as demonstratedby trypsin pre-treatment of purified PBCV-1 virus particles. d, Northernblot analysis establishes that vset is expressed as an ˜1.8 kDatranscript in PBCV-1 infected Chlorella NC64A cells beginning at 60minutes after infection. e, Histone H3K27 methylation activity of vSETin the lysate of PBCV-1 virions as demonstrated using western blotanalysis with anti-H3-K27me2 antibodies.

FIGS. 2a-2i show the methylation of Chlorella histone H3-K27 by vSET. a,Sequence alignment of H3 from Chlorella NC64A, Homo sapien, Musmusculus, Xenopus laevis, Caenorabditis elegans and Chlamydomonasreinhardtii. Residues different in H3 are shown in blue. b, vSETmethylates H3 in nucleosomes. Native histone H3 was used as a control(right lane). c, HKMTase activity of vSET targets H3-K27, as illustratedby methyl transfer from ¹⁴C-methyl-SAM to GST-fusion H3 peptides(residues 1-57). Fluorescent values relative to wild-type enzymeactivity are indicated under the lower panel. Relative amounts of GST-H3in the assays are shown in SDS-PAGE (upper panel). d, Western blotanalysis of vSET-treated GST, GST-H3 wild-type and K27R mutant peptidesusing anti-H3-K27me1/2/3, anti-H3-K4me2, anti-H3-K9me2 andanti-H3-K36me2 antibodies. e, Western blot analyses of Chlorella H3before and after PBCV-1 infection at specified time points usingantibodies against histone H3 with different modifications. f, vSET islocalized in the nucleus of PBCV-1 infected Chlorella cells collected 60minutes after infection, as illustrated by immuno-fluorescence.Uninfected and 60 min post-infected Chlorella cells were probed withanti-vSET (upper and middle panels, respectively) or anti-H3 antibodies(lower panel). g, vSET and host Chlorella H3-K27me2 co-localize in thenucleus 60 minutes post PBCV-1 infection, as shown by confocalfluorescence imaging. Chlorella cells were probed with anti-vSET andanti-H3-K27me2 antibodies and visualized by immunofluorescence usingAlexa488 and Alexa594 dyes, respectively. h-i show the enzyme kineticsof H3-K27 methylation by vSET, as determined using a H3 peptide (aa13-33) substrate and mass spectrometry analysis: h, K27 methylation of aH3 peptide from unmodified state to mono-, di- and tri-methylationstates catalyzed by vSET as illustrated by mass spectrometry analysis atspecified time points during the reaction; i, The entire time course ofthe H3-K27 methylation of the H3 peptide by vSET as analyzed by massspectrometry.

FIGS. 3a-3d show the nuclear localization and H3-K27 methylation ofH3-K27 by vSET. a, Nuclear localization of GFP-vSET in transientlytransfected NIH-3T3 cells. Fluorescence images show that wild-type vSETis located in the nucleus, whereas a triple mutant, KR(M)R(SEQ IDNO:3)/AA(M)A (SEQ ID NO:4) (residues 85-88) is in the cytoplasm. Cellswere counterstained with DAPI to highlight nuclei. b, Western blotanalysis of Hela cells showing effects of treatment of EZH2-specific andmock siRNAs on protein expression of EZH2, RING1, SUZ12 and histone H3with different modifications. Lamin B was used as a control. c, Westernblot analysis of the nuclear extract of Hela cells treated with EZH2siRNA and subject to in vitro methylation by vSET. d, Western blotanalysis showing di and tri-methylation of H3-K27 upon tetracyclineinduction of stably transfected vSET in Hela cells 72 hours after EZH2knock-down by siRNA.

FIGS. 4a-4j show targeted and global gene transcription repression byvSET via its histone H3-K27 methylation activity. a, Transcriptionrepression of a reporter luciferase gene by vSET and its active sitemutant in transiently transfected 293T cells, in which vSET was fused tothe Ga14-DNA binding domain. The error bars represent the standarddeviation of triplicate assays. b, Transcription repression of thereporter luciferase gene by Flag- or HA-tagged vSET and its active sitemutant in transiently transfected 293T cells. This assay shows theglobal effect of the luciferase repression by vSET H3-K27 methylationactivity, as vSET is not fused Gal-DBD as in a. c, vSET repression ofTat-mediated transcription of a HIV LTR-luciferase reporter gene intransfected HeLa cells. d, CHIP analysis of vSET repression ofTat-mediated transcription of the HIV LTR-luciferase reporter gene intransfected Hela cells on the HIV promoter sequence in Nuc2 usingvarious antibodies against histone H3 with different modifications.GADPH and IgG were used as control. e, Western blot analyses ofassociation of the Polycomb group repression complexes 1 and 2 (PRC1 andPRC2) proteins with H3-K27 of different modifications upon induction ofstably transfected vSET in HeLa cells with EZH2 knock-down by siRNA. Theinactive vSET-Y105F mutant was used as control. f, Quantitative RT-PCRmeasurements showing relative mRNA levels of five Polycomb target genesof Hey1, HOXA9, HOXD8, HOXB9, and HOXA7, and three house keeping genesof GAPDH, RPS, and Tubulin in normal HeLa cells, and EZH2 knocked-downHeLa cells with or without induction of the transiently transfectedvSET, as well as HeLa cells transfected with vSET. g, vSET induces G2/Mphase cell accumulation in NIH-3T3 cells transiently co-transfected witha vSET-encoding pCMV-tag2B vector and a marker Us9-GFP-encoding plasmid.Western blots showing relative equal expression of vSET and its mutants.h, vSET expression induces G2/M arrest in stably transfected HeLa cellswith tetracycline control. The DNA content of the gated GFP-positivecells was determined by PI staining and FACS analysis. Average values ofthe DNA content of cell cycle phases represent at least threeindependent transfection experiments. i, Repression of HOXA7 activationin the HeLa cells with or without EZH2 knockdown by tetracycline inducedvSET in a luciferase assay. j, ChIP analysis at the HOXA7 promotor uponinduction of vSET in the HeLa cells with and without EZH2 siRNAtreatment.

FIGS. 5a-5d show HKMT activity of SET proteins from chlorella viruses.a, Southern dot blot analysis of 37 chlorella viruses confirmed thepresence of a vset-like gene in 32 viruses. vset-like genes from 3 ofthe 5 viruses that did not exhibit positive hybridization signals, i.e.NY-2A, NY-2B and MA-1D were detected using low stringency Southernhybridization. b, Sequence alignment of vSET and SET proteins fromchlorella viruses NY2-A, NY-2B and MA-1D. Positions of α helices and βstrands in the vSET sequence are shown as indicated. Residues absolutelyconserved among all known SET proteins are indicated by a (●) above theresidues. Residues in the three viral SET proteins that differ from vSETare also shown. Residues of vSET at the dimer interface are underlined.The NLS sequence in PBCV-1 is boxed. c, Methylation activity of the SETproteins from viruses NY-2A, NY-2B and MA-1D measured with H3 peptidesusing ¹⁴C-methyl-SAM. d, HMT activity of the viral SET proteins usingwild-type H3 (residues 1-57) versus the K27R mutant. The fluorogramshows that NY-2A, NY-2B and MA-1D SET proteins methylate wild-type H3but not the K27R mutant. H3 modified by vSET served as a control.

FIGS. 6a-6f show effects of vSET histone H3-K27 methylation ontranscription repression. a, Optimization of concentration of histoneH3-K27 mono-, and di- and tri-methylation specific antibodies to be usedin Western blot analysis. b, Western blot analyses of various H3modification-specific antibodies using native H3 isolated from calfthymus (Roche). c, Western blot analyses of association of the Polycombgroup repression complexes 1 and 2 (PRC1 and PRC2) proteins with histoneH3-K27 of different modifications upon induction of stably transfectedvSET in HeLa cells. Lamin B was used as control. d, e, vSET repressionof Tat-mediated transcription of a HIV LTR-luciferase reporter gene intransfected 293T cells, or HeLa cells, respectively, in a dose dependentmanner. The error bars represent the standard deviation of triplicateassays. f, tetracycline induction of vSET in the HeLa cells with andwithout EZH2 knockdown by siRNA.

FIGS. 7a-7c show that a triple mutation in vSET, KR(M)R (SEQ ID NO:3)-to-AA(M)A (SEQ ID NO:4) (aa 85-88), does not disrupt the overall foldof the protein or its HKMTase activity. a, GST-fusion H3 (residues1-57), wild-type and the K27R mutant, were used as substrates in an invitro HKMTase assay and the relative amounts of GST-H3 used in the assayare shown in an SDS-PAGE gel (upper panel). Signals in the fluorogramindicate that similar to wild-type vSET, the triple mutant, methylatedwild-type GST-H3 (aa 1-57) but not the K27R mutant. This indicates thatthe triple mutation in vSET did not affect the enzyme activity andspecificity of vSET for H3-K27. b & c, Gel filtration chromatographyverified that the triple mutation of vSET did not affect the overallfold of the protein. Hexa-histidine tagged vSET, wild-type and mutant,eluted as about 30 kDa proteins, which is the approximate value of ahexahistidine tagged vSET dimer, MW of 31.6 kDa. The protein standardsused were ribonuclease A (14 kDa), chymotrypsin (25 kDa) and ovalbumin(43 kDa).

FIG. 8 shows that expression of vSET in Arabidopsis leads to apoptosis.Transgenic plants that contain a beta-estradiol inducible vectorexpressing vSET. a. Arabidopsis before induction of vSET expression.b,c. Two Arabidopsis plants 10 days after beta-estradiol inducedexpression of vSET.

FIG. 9 shows the nucleic acid sequence of the PBCV-1 gene A612L thatencodes the vSET protein.

FIG. 10 shows the amino acid sequence of the vSET polypeptide encoded bythe PBCV-1 gene A612L.

FIG. 11 shows the transcriptional silencing of disease related genes byvSET.

FIG. 12 shows histone H3K27 methylation by vSET. Western blot analysesof H3 modification-specific antibodies using synthesized histonepeptides containing specific modified amino acids as stated.

FIGS. 13a-13d show Histone H3K27 as the major substrate by vSET. a, EZH2knockdown in HeLa cells by RNAi and loss of dimethylation at histoneH3K27. b, Strategy for the EZH2 RNAi experiment. c, SDS-PAGE and westernblot analysis of cellular extracts of EZH2-knockdown HeLa cells andchlorella cells using H3, H3K27me2, H3K9me2, and H3K4me, and Pan-Kmeantibodies. d, Broad methylated-lysine specific recognition by thePan-Kme antibody.

FIGS. 14a-14d show full scans of key western blots of FIGS. 1B, 2D, 2E,3D, 4A, 4B and 4E. Arrowheads show specific bands in cropped images.

FIG. 15 shows a table of the antibodies in the experiments described inthe application.

FIG. 16 shows tables describing the amino acid sequences of histone H3Peptides (aa 1-57) used in FIG. 2C; the sequences of siRNAs used forHuman EZH2 Knockdown experiments; and a list of RTPCR and ChIP Primersused in the experiments described in the application.

FIG. 17 shows a schematic representation of the luciferase reporter geneconstructs used in the experiments of the application as described inFIGS. 4a and 4 b.

DETAILED DESCRIPTION

The present invention provides for selective or general suppression orinhibition of gene expression. Suppression of gene expression is apowerful tool for treating a number of disease states associated withinappropriate gene expression, particularly cancer. While a number ofapproaches are in development, including interfering RNA (RNAi),antisense, histone deacetylase inhibitors, and the use of intercalatingagents, and pseudo-expression blockers that actually target theexpressed protein, the present invention represents a new approach.

The invention is based, in part, on the discovery that viruses candirectly modify host chromatin to interfere with host genetranscription. Particularly, it was discovered that Paramecium bursariachlorella virus 1 (PBCV-1) encodes a functional SET domain histonelysine methyltransferase (termed vSET) that is directly linked to rapidinhibition of host transcription after virus infection. Additionally, itwas discovered that vSET is packaged in the PBCV-1 virion and comprisesa nuclear localization signal. vSET causes host transcriptionalrepression by selective methylation of histone H3 at lysine 27, apost-translational modification that is well established to triggerlong-term gene silencing in eukaryotes. Further, vSET induces cellaccumulation at the G2/M phase of the cell cycle by recruiting thePolycomb repressive complex 1 (PRC1) wherein the PRC1 component proteinCBX8 binds to the methylated H3-K27 in mammalian cells. Finally, it wasdiscovered that vSET-like proteins exhibiting H3-K27 methylationactivity are conserved in the chlorella virus family.

Certain experimental results underlie the present invention:transfection of human embryonic kidney 293T cells with wild type vSETsuppressed gene transcription, resulting in about 60-90% transcriptionrepression in the host cell.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them.

As used herein, the term “gene expression” refers to the transcriptionof RNA from a genomic DNA (gDNA) template. The RNA can be messenger RNA(mRNA), which is subsequently translated on ribosomes to produceprotein; ribosomal RNA (rRNA); or transfer RNA (tRNA). The latter twoforms of RNA are involved in the synthesis of all cellular proteins.“Gene expression” may also refer to synthesis of a protein.

As used herein, “suppression” or “inhibition” of gene expression refersto a reduction in the normal level of expression.

The cell in which target gene expression is suppressed/inhibited may beany eukaryotic cell. In specific embodiments, the cell may be an animalcell, a plant cell, a mammalian cell, a human cell, a murine cell, aracine cell, a canine cell, a feline cell, an equine cell, a bovinecell, an ovine cell, a porcine cell, etc.

A “multicellular organism” can be any animal or plant. In specificembodiments, the multicellular organism is a mammal, including but notlimited to the human, canine, feline, equine, bovine, ovine, porcine,murine, racine, etc.

The term “therapeutically effective amount” refers to the amount of theintroduced protein that is sufficient to result in a therapeuticresponse. A therapeutic response may be any response that a user (e.g.,a clinician) will recognize as an effective response to the therapy,including symptoms and surrogate clinical markers described herein.Thus, a therapeutic response will generally be an amelioration of one ormore symptoms or signs of a disease or disorder, or an increasedsurvival, for example, but not limited to, a decrease in thetranscriptional expression of an oncogenic gene, an inflammatory genesuch as a cytokine, a transcription factor gene, a developmental gene,or a viral replication gene.

The term “effective amount” refers to an amount of introduced proteinthat is either a therapeutically effective amount or an amount that issufficient to suppress or inhibit expression of a targeted gene.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce untoward reactions when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans.

The term “chromatin histone protein” has its ordinary meaning, e.g., asset forth in Jenuwein et al., Science, 293:1074-1080 (2001); Cheung etal., Cell, 103: 263-271 (2000); Turner et al., Cell, 111:285-291,(2002); Kouzarides et al., Curr. Opin. Genet. Dev., 12: 198-209 (2002);and Lachner et al., Curr. Opin. Cell Biol., 14:286298 (2002)

Lysine Methyltransferase

In one non-limiting embodiment of the invention, the protein introducedaccording to the present invention is a lysine methyltransferase. Theterm “lysine methyltransferase” refers to a polypeptide which exhibitsmethyltransferase activity. The polypeptide can transfer one, two, orthree or more methyl groups from a methyl donor to a lysine residuewithin the intracellular environment of a cell. Alternatively, thelysine methyltransferase can transfer one, two, or three or more methylgroups from a methyl donor to a lysine residue in an extracellularenvironment, for example, in an in vitro cell-free translation systems.Methyltransferases are described generally in, for example, Nightingaleet al., Curr Opin Genet Dev 16, 125-36 (2006); Fischle et al., Curr OpinCell Biol 15, 172-83 (2003); Bannister et al., Cell 109, 801-6 (2002);Lachner et al., J Cell Sci 116, 2117-24 (2003); Sims et al., Genes Dev20, 2779-86 (2006); Wysocka et al., Nature 442, 86-90 (2006); andBernstein et al., Proc Natl Acad Sci USA 99, 8695-700 (2002), which arehereby incorporated by reference in their entireties.

In accordance with the present invention, the lysine methyltransferasehas the properties of a histone lysine methyltransferase. In a specificembodiment, the protein is a histone lysine methyltransferase. In oneembodiment, the histone lysine methyltransferase is an enzyme that isfunctional when present as a dimer, wherein the protein can function totransfer one, two, or three or more methyl groups from a methyl donor toa lysine residue comprised in a histone protein.

The methyl donor may be any agent which comprises one or more methylgroups, such as, for example but not limited to,S-adenosyl-L-methionine. The methyl donor may be endogenous to the cellor organism to which the lysine methyltransferase is introduced.Alternatively, the methyl donor is introduced onto the cell or organismbefore, at the same time, or after the lysine methyltransferase isintroduced.

In a specific embodiment of the invention, lysine methyltransferase is aviral histone lysine methyltransferase that can transfer a methyl groupfrom a methyl donor to a lysine residue of a chromatin histone protein.In a further embodiment, the viral histone lysine methyltransferasetransfers a methyl group to Lysine 27 of the chromatin histone 3 protein(H3-K27). The H3-K27 may be mono-, di-, or tri-methylated by the histonelysine methyltransferase. In a further embodiment, the viral histonelysine methyltransferase comprises an SET domain.

In a preferred embodiment of the invention, the lysine methyltransferaseof the present invention is a protein comprising a Chlorella virus SETdomain of a viral histone lysine methyltransferase.

In one embodiment exemplified below, the lysine methyltransferase of thepresent invention is a viral histone lysine methyltransferase comprisingan SET domain from Paramecium bursaria chlorella virus 1 (vSET). Theviral histone lysine mehtyltransferase is preferably encoded by theParamecium bursaria chlorella virus 1 (PBCV-1) gene A612L, a nucleicacid which encodes the PBCV-1 vSET histone lysine methyltransferase(Nucleotides 293003-293362 of GenBank accession number NC_000852) (SEQID NO:1).

In another non-limiting embodiment, the viral histone lysinemehtyltransferase can be encoded for by a vset-like gene, a nucleic acidisolated from any chlorella virus which encodes a vSET-like protein thatcan function as a methyltransferase, specifically, a protein that canmethylate H3-K27. Alternatively, the viral histone lysinemethyltransferase can be encoded by any nucleic acid molecule exhibitingat least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or up to 100% homology to the A612L gene (where homology maybe determined by standard software such as BLAST or FASTA), and anysequences which hybridize under standard conditions to these sequences.

In another non-limiting embodiment, the viral histone lysinemethyltransferase may be any other protein or may be encoded by anyother nucleic acid sequence that encodes a viral histone lysinemethyltransferase (having the same functional properties as theaforementioned polypeptide sequences) that have the ability to transferone, two, or three or more methyl groups from a methyl donor to thehistone, e.g., H3-K27.

In a specific embodiment exemplified below, a vSET histone lysinemethyltransferase also refers to an amino acid sequence depicted in SEQID NO:2 (GenBank accession number AAC96946), and any other amino acidsequence that encodes a viral histone lysine mehtyltransferasepolypeptide having the same methyltransferase function of SEQ ID NO:2and which may have at least about 80%, at least 90%, at least 95%, or upto 100% homology thereto.

In one embodiment the viral histone lysine methyltransferase is a vSETpolypeptide that is purified from Paramecium bursar chlorella virus 1.Alternatively, the viral histone lysine methyltransferase is a vSET-likepolypeptide purified from any other virus of the chlorella virus familyincluding, for example, but not limited to, chlorella viruses NE-8D,NYb-1, CA-4B, AL-1A, NY-2C, NC-1D, NC-1C, CA-1A, CA-2A, IL-2A, IL-2B,IL-3A, IL-3D, SC-1A, SC-1B, NC-1A, NE-8A, AL-2C, MA-1E, NY-2F, CA-1D,NC-1B, NY-s1, IL-5-2s1, AL-2A, MA-1D, NY-2B, CA-4A, NY-2A, XZ-3A, SH-6A,BJ-2C, XZ-6E, XZ-4C, XZ-5C, and XZ-4A.

Alternatively, the viral histone lysine methyltransferase is arecombinant vSET polypeptide encoded by a recombinant nucleic acid, forexample, a recombinant DNA molecule. The nucleic acid can encode any ofthe specific vSET or vSET-like proteins identified above.

In one non-limiting embodiment, the lysine methyltransferase comprises anuclear localization signal. As used herein, “nuclear localizationsignal” refers to a molecule or polypeptide that facilitates themovement of the lysine methyltransferase to the nucleus of the cell towhich the lysine methyltransferase is introduced. The nuclearlocalization signal can be any agent or compound that facilitates themovement of the lysine methyltransferase to the nucleus of a cell wherethe enzyme transfers one or more methyl groups from a methyl donor to atarget gene. Examples of nuclear localization signals include, but arenot limited to, polypeptides such as, for example, the amino acidsequences Lys-Arg-Met-Arg (KRMR) (SEQ ID NO:3), Pro-Arg-Ile-Val (PRIV)(SEQ ID NO:5), Lys-Arg-Pro-Arg (KRPR) (SEQ ID NO:6), and Arg-Arg-Pro-Arg(RRPR) (SEQ ID NO:7).

Targeted Methyl Transferases

In another, non-limiting embodiment, the lysine methyltransferasefurther comprises a targeting protein, such as a DNA binding protein,which increases the specificity of methylation relative to the targetedgene or protein associated therewith. The lysine methyltransferase maybe fused to the targeting protein in the form of a fusion protein, or,alternatively, an expression vector may encode a lysinemethyltransferase fused to a targeting protein. As used herein, “DNAbinding protein” means a protein that contains a polypeptide sequencethat specifically interacts with DNA regulatory sequences associatedwith a gene or class of genes. For purposes of this invention, the wholeprotein or just the DNA binding domain of the protein can be a “DNAbinding protein.” Such DNA binding proteins include transcriptionfactors, enhancer proteins, suppressor proteins, and the like.Non-limiting examples of DNA binding protein are NF-kB and DNA bindingdomains from the polycomb target genes HOXC6 and HOXC8.

The DNA binding protein is effective to target the lysinemethyltransferase to one or more specific “targeted genes,” thus thetargeted gene is methylated while non-targeted genes remainsubstantially unaffected by the lysine methyltransferase. Therefore onlythe targeted genes experience transcriptional expression suppression.The methods of the present invention contemplate the targeting of anygene for transcription suppression.

In one non-limiting embodiment, genes that are targeted fortranscription suppression are genes involved in oncogenesis and/ortumorigenesis. As used herein, the term “oncogenesis” refers to theprogression of cytological, genetic, and/or cellular changes thatculminate in a malignant tumor. As used herein, the term “tumorigenesis”refers to the abnormal growth of tissue resulting in a swelling or massof tissue. For example, targeted genes include inhibitors of tumorsuppressors, such as murine double minute 2 (MDM2); oncogenic proteins,such as Src tyrosine kinases, Ras kinases, receptor tyrosine kinases,epidermal growth factor receptor (EGFR), platelet-derived growth factorreceptors (PDGFR), and vascular endothelial growth factor receptors(VEGFR); transcription factors such as myc or NF-kB; inflammatoryfactors and cytokines such as tumor necrosis factor-alpha (TNF-α) ORNF-kB, transforming growth factor-beta (TGF-β), interferon-gamma(IFN-γ), interleukin-2 (IL-2) and interleukin-10 (IL-10); homeodomaingenes such as HOXA2, HOXA5, HOXA7, HOXA9, HOXB9, HOXC6, HOXC8, HOXD8,and Hey1; receptors such as Androgen Receptor, Retinoic Acid receptor(RAR), or Retinoic Acid X receptor (RXR); cell cycle regulating proteins(e.g., proteins that regulate cell division) such as Cyclin D; and viralreplication factors such as human immunodeficiency virus type 1transactivator protein (tat).

Targeted gene expression suppression according to the instant inventiontherefore provides for methods of treating a disease or disorder inwhich suppression of gene expression can provide a therapeutic benefit.Such diseases or disorders include, but are not limited to, prostatecancer (androgen receptor and/or HOXC8), developmental disorders(HOXA2), breast cancer (HOXA5 and/or E Cadherin), Ovarian cancer (HOXA7and/or E Cadherin), blood disorders (HOXA9), Leukemia (Retinoic Acidreceptor (RAR)), cancer (Retinoic Acid X receptor (RXR), and/or M50/BetaCatenin, and/or Cyclin D), basal cell carcinoma (M50/Beta Catenin), andinflammatory disorders (NF-κB).

In another non-limiting embodiment, the genes that are targeted fortranscription suppression are regulatory genes which function tosuppress the expression of a second gene or genes. Targeting theregulatory genes, according to the present invention, reduces thesuppression of the second gene, thus, increasing the second gene'sexpression.

In one embodiment, the second gene is, for example, but not by way oflimitation, a transmembrane protein which functions, for example, incell adhesion and/or tumor suppression. Such proteins include, forexample, E cadherin or M50/Beta-catenin. Increasing the expression of asecond gene as described above provides for methods of treating adisease or disorder in which transcriptional activation of geneexpression can provide a therapeutic benefit. Such diseases or disordersinclude, but are not limited to, cancers. For example, loss of functionor reduced expression of E-cadherin, a tumor suppressor gene, is thoughtto contribute to progression of cancer (such as, but not limited to,gastric, breast, colorectal, thyroid and ovarian cancer), by increasingproliferation, invasion, and/or metastasis. Therefore, transcriptionactivation of E-cadherin by vSET, through a reduction in expression ofan E-cadherin suppressor, can provide a therapeutic benefit.

In another non-limiting example, the expression of Beta-catenin isincreased via a vSET mediated reduction in expression of a Beta-cateninsuppressor. Beta-catenin plays an important role in Wnt signalingpathway, for example, in various aspects of liver biology includingliver development (both embryonic and postnatal), and liver regenerationfollowing partial hepatectomy (Thompson et al., Hepatology45(5):1298-305 (2007)).

In another non-limiting example, the expression of Retinoic Acid Xreceptor (RXR) is increased via a vSET mediated reduction in expressionof an RXR suppressor. Increased expression of RXR can have beneficialeffects on vision, immune function, bone metabolism, skin health, andreducing risk of heart disease.

In another embodiment, the targeted gene is a regulatory gene whichpromotes the expression of a second gene. Targeting the regulatory geneaccording to the present invention reduces the expression of the secondgene.

Suppression or Inhibition of Gene Expression

A lysine methyltransferase can be introduced into a cell of amulticellular organism through a variety of techniques. It can beintroduced as a protein or through an expression vector that producesthe protein in the cell in situ.

When introduced into a cell, unmodified lysine methyltransferaseexhibits non-specific methylation of a cell's genes, thus suppressingthe transcriptional expression of at least one, two, three, four or upto all of the genes in the cell. Targeted lysine methyltrasnferaseexhibits specific methylation at the target gene (or genes sharing thesame target characteristics), thus suppressing that gene (or genes).

In one non-limiting embodiment of the invention, methylation of a targetgene's chromatin H3-K27 by a lysine methyltransferase suppresses thetranscription of the gene or genes. The transcription suppression of thegene or genes can be between about 5% and 100%, more preferably betweenabout 20% and 95%, more preferably between about 40% and 85%, and mostpreferably between about 60% and 90% suppression of target genetranscription as compared to a wild-type target gene that is notmethylated at H3-K27. Where multiple genes are targeted, not every genewill necessarily be suppressed to the same degree.

In one embodiment, the lysine methyltransferase may be administered witha histone demethylase inhibitor. The histone demethylase inhibitor maybe administered at the same time as, before, or after administration ofthe lysine methyltransferase.

In one embodiment, the transcription suppression can occur from about 1to about 120 minutes, more preferably from about 5 to about 60 minutes,more preferably from about 10 to about 50 minutes, and most preferablyfrom about 20 to about 40 minutes following introduction of the lysinemethyltransferase into a cell containing the target gene.

In a further non-limiting embodiment, when the methylation of a targetgene's chromatin H3-K27 by the lysine methyltransferase occurs in acell, the methylation results in arrest of the cell cycle. Methylationcan result in accumulation of cells at the cell cycle phase in whicharrest occurs. For example, NIH-3T3 or HeLa cells transientlytransfected with vSET results in arrest of the cell cycle at the G2/Mtransition, and accumulation of cells at the G2/M cell cycle phase.

Methylation of H3-K27 by vSET can result in long term gene transcriptionsuppression that can persist for up to 1 hour, 1 day, or longer.Alternatively, the gene transcription suppression can be permanent, forexample, suppression of gene transcription in a cell can persist as longas the cell is alive. Long term gene transcription suppression can beachieved through recruitment of proteins to the methylated H3-K27 site.For example, following methylation by a viral histone lysinemethyltransferase, H3-K27 can become associated with PcG PRC1 orEzh2/PRC2 complexes that lead to long-term gene silencing. In onespecific, non-limiting embodiment, the PcG PRC1 protein CBX8 becomesassociated with di-methylated and tri-methylated H3-K27, resulting inlong term gene silencing.

In another non-limiting embodiment of the invention, the lysinemethyltransferase is administered to a cell with a histone demethylaseinhibitor. Such inhibitors include, for example, phenelzine,tranylcypromine, nialamide, clorgyline, deprenyl, and pargyline.

Delivery of Proteins

According to the invention, the methylating protein may be introducedeither as a protein or as a nucleic acid encoding the protein. Where themethylating protein is provided as a protein, numerous methods can beemployed to achieve uptake and targeting of the lysine methyltransferaseby the cells. According to the invention, the lysine mehtyltransferasedelivered to a cell or administered to an organism can be a proteinisolated from a cell culture, for example, a eukaryotic cell line, inwhich the cell culture has been infected with a chorella virus (e.g.,PBCV-1). Alternatively, the lysine methyltransferase can be arecombinant protein. Peptide sequences have been identified that mediatemembrane transport, and accordingly provide for delivery of polypeptidesto the cytoplasm. For example, such peptides can be derived from theAntennapedia homeodomain helix 3 to generate membrane transport vectors,such as penetratin (see PCT Publication WO 00/29427; see also Fischer etal., J. Pept. Res. 55:163-72 (2000); DeRossi et al., Trends in CellBiol. 8:84-7 (1998); Brugidou et al., Biochem. Biophys. Res. Comm.214:685-93 (1995)), the VP22 protein from herpes simplex virus (Phelanet al., Nat. Biotechnol. 16:440-3 (1998)), and the HIV TATtranscriptional activator. Protein transduction domains, including theAntennapedia domain and the HIV TAT domain (see Vives et al., J. Biol.Chem. 272:16010-17 (1997)), possess a characteristic positive charge,which led to the development of cationic 12-mer peptides that can beused to transfer therapeutic proteins and DNA into cells (Mi et al.,Mol. Therapy 2:339-47 (2000)). The above-mentioned protein transductiondomains are covalently linked to the target protein, either by chemicalcovalent cross-linking or generation as a fusion protein. Further, anon-covalent, synthetic protein transduction domain has been developedby Active Motif Inc. (Carlsbad, Calif.). This domain associates with thetarget protein through hydrophobic interactions, and advantageouslydissociates from the protein once inside the cell (Morris et al., Nat.Biotechnol. 19:1173-6 (2001)). In addition, lipid carriers have recentlybeen shown to deliver proteins into cells in addition to an establisheduse for delivering naked DNA (Zelphati et al., J. Biol. Chem.276:35103-10 (2001)). For an overview of protein translocationtechniques see Bonetta, The Scientist 2002; 16(7):38.

Gene Therapy

The term “gene therapy” refers to a method of changing the expression ofan endogenous gene by exogenous administration of a second gene. As usedherein, gene therapy also refers to the replacement of defective geneencoding a defective protein, or replacement of a missing gene, byintroducing a functional gene corresponding to the defective or missinggene into somatic or stem cells of an individual in need.

The gene to be administered for the methods of the present invention canbe isolated and purified using ordinary molecular biology, microbiology,and recombinant DNA techniques within the skill of the art. For example,nucleic acids encoding the target protein can be isolated usingrecombinant DNA expression as previously described, and as described inthe literature. See, e.g., Sambrook et al. (2001) Molecular Cloning: ALaboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press: ColdSpring Harbor, N.Y., which is incorporated herein by reference in itsentirety. The nucleic acid encoding the protein may be full-length ortruncated, so long as the gene encodes a biologically active protein.

The identified and isolated gene can then be inserted into anappropriate cloning vector. Vectors suitable for gene therapy includeviruses, such as adenoviruses, adeno-associated virus (AAV), vaccinia,herpesviruses, baculoviruses and retroviruses, parvovirus, lentivirus,bacteriophages, cosmids, plasmids, fungal vectors and otherrecombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

In a preferred embodiment, the vector is a viral vector. Viral vectors,especially adenoviral vectors can be complexed with a cationicamphiphile, such as a cationic lipid, polyL-lysine (PLL), anddiethylaminoethyldextran (DELAE-dextran), which provide increasedefficiency of viral infection of target cells (See, e.g., U.S. Pat. No.5,962,429, incorporated herein by reference).

The coding sequences of the gene to be delivered are operably linked toexpression control sequences, e.g., a promoter that directs expressionof the gene. As used herein, the phrase “operatively linked” refers tothe functional relationship of a polynucleotide/gene with regulatory andeffector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signalsequences. For example, operative linkage of a nucleic acid to apromoter refers to the physical and functional relationship between thepolynucleotide and the promoter such that transcription of DNA isinitiated from the promoter by an RNA polymerase that specificallyrecognizes and binds to the promoter, and wherein the promoter directsthe transcription of RNA from the polynucleotide. Furthermore, theexpression vector can comprise inducible promoters, for example, abeta-estradiol inducible expression vector. Inducible expression vectorsare well known in the art. In one non-limiting example, the expressionvector is a viral vector, for example, lenti-virus vector pLVET-tTRKRABor adeno virus vector, VQpacAd5CMVK-NpA (Viral Quest).

In one specific embodiment, a vector is used in which the codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for expression of the construct from a nucleic acid moleculethat has integrated into the genome (Koller and Smithies, Proc. Natl.Acad. Sci. USA, 86:8932-8935 (1989); Zijlstra et al., Nature,342:435-438 (1989); U.S. Pat. No. 6,244,113 to Zarling et al.; and U.S.Pat. No. 6,200,812 to Pati et al.), each of which are herebyincorporated by reference in their entirety.

Delivery of Gene Therapy Vectors

Where the methylating protein is introduced via a nucleic acid, saidnucleic acid may be comprised in a gene therapy vector. Delivery of agene therapy vector into a patient may be either direct, in which casethe patient is directly exposed to the vector or a delivery complex, orindirect, in which case, cells are first transformed with the vector invitro, then transplanted into the patient. These two approaches areknown, respectively, as in vivo and ex vivo gene therapy.

Direct Transfer.

In a specific embodiment, the vector is directly administered in vivo,where it enters the cells of the organism and mediates expression of thegene. This can be accomplished by any of numerous methods known in theart, e.g., by constructing it as part of an appropriate expressionvector and administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector, or by direct injection of naked DNA, or by use of microparticlebombardment; or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in polymers or biopolymers,encapsulation in liposomes, microparticles, or microcapsules, byadministering it in linkage to a peptide or other ligand known to enterthe nucleus; or by administering it in linkage to a ligand subject toreceptor-mediated endocytosis, etc. In another embodiment, a nucleicacid-ligand complex can be formed in which the ligand comprises afusogenic viral peptide to disrupt endosomes, allowing the nucleic acidto avoid lysosomal degradation, or cationic 12-mer peptides, e.g.,derived from antennapedia, that can be used to transfer therapeutic DNAinto cells (Mi et al., Mol. Therapy 2:339-47 (2000)). In yet anotherembodiment, the nucleic acid can be targeted in vivo for cell specificuptake and expression, by targeting a specific receptor (see, e.g., PCTPublication Nos. WO 92/06180, WO 92/22635, WO 92/20316 and WO 93/14188).Additionally, a technique referred to as magnetofection can be used todeliver vectors to mammals. This technique associates the vectors withsuperparamagnetic nanoparticles for delivery under the influence ofmagnetic fields. This application reduces the delivery time and enhancesvector efficacy (Scherer et al., Gene Therapy 9:102-9 (2002)).

In a specific embodiment, the nucleic acid can be administered using alipid carrier. Lipid carriers can be associated with naked nucleic acids(e.g., plasmid DNA) to facilitate passage through cellular membranes.Cationic, anionic, or neutral lipids can be used for this purpose.However, cationic lipids are preferred because they have been shown toassociate better with DNA which, generally, has a negative charge.Cationic lipids have also been shown to mediate intracellular deliveryof plasmid DNA (Felgner and Ringold, Nature 1989; 337:387). Intravenousinjection of cationic lipid-plasmid complexes into mice has been shownto result in expression of the DNA in lung (Brigham et al., Am. J. Med.Sci. 1989; 298:278). Representative cationic lipids include thosedisclosed, for example, in U.S. Pat. No. 5,283,185; and e.g., U.S. Pat.No. 5,767,099, the disclosures of which are incorporated herein byreference.

Preferably, for in vivo administration of viral vectors, an appropriateimmunosuppressive treatment is employed in conjunction with the viralvector, e.g., adenovirus or lentivirus vector, to avoidimmuno-deactivation of the viral vector and transfected cells. Forexample, immunosuppressors such as anti-CD4 antibody, can beadministered to block humoral or cellular immune responses to the viralvectors. In that regard, it is advantageous to employ a viral vectorthat is engineered to express a minimal number of antigens.

Indirect Transfer.

Somatic cells may be engineered ex vivo with a construct encoding aprotein, for example, a lysine methyltransferase, and re-implanted intoan individual. This method is described generally in WO 93/09222 toSelden et al., which is hereby incorporated by reference in itsentirety. In addition, this technology is used in Cell Based Delivery'sproprietary ImPACT technology, described in Payumo et al., Clin.Orthopaed. and Related Res. 403S: S228-S242 (2002). In such a genetherapy system, somatic cells (e.g., fibroblasts, hepatocytes, orendothelial cells) are removed from the patient, cultured in vitro,transfected with the gene(s) of therapeutic interest, characterized, andreintroduced into the patient. Both primary cells (derived from anindividual or tissue and engineered prior to passaging), and secondarycells (passaged in vitro prior to introduction in vivo) can be used, aswell as immortalized cell lines known in the art. Somatic cells usefulfor the methods of the present invention include but are not limited tosomatic cells, such as fibroblasts, keratinocytes, epithelial cells,endothelial cells, glial cells, neural cells, formed elements of theblood, muscle cells, other somatic cells that can be cultured, andsomatic cell precursors. In a preferred embodiment, the cells arefibroblasts or mesenchymal stem cells.

Nucleic acid constructs, which include the exogenous gene and,optionally, nucleic acids encoding a selectable marker, along withadditional sequences necessary for expression of the exogenous gene inrecipient primary or secondary cells, are used to transfect primary orsecondary cells in which the encoded product is to be produced. Suchconstructs include but are not limited to infectious vectors, such asretroviral, herpes, adenovirus, lentivirus, adenovirus-associated, mumpsand poliovirus vectors, can be used for this purpose.

Transdermal delivery is especially suited for indirect transfer usingcell types of the epidermis including keratinocytes, melanocytes, anddendritic cells (see, e.g., Pfutzner et al., Expert Opin. Investig.Drugs 9:2069-83 (2000), which is hereby incorporated by reference in itsentirety).

Mesenchymal stem cells (MSCs) are non-blood-producing stem cellsproduced in the bone marrow. MSCs can be made to differentiate andproliferate into specialized non-blood tissues. Stem cells transfectedwith retroviruses are good candidates for the therapy due to theircapacity for self-renewal. This ability precludes repetitiveadministration of the gene therapy vector. Another advantage is that ifthe injected stem cells reach the target organ and then differentiate,they can replace the damaged or malformed cells at the organ

Control of Gene Expression for Disease Therapy

In one embodiment, the present invention provides for methods ofsuppressing the transcriptional expression of one or more genes in acall by introducing an effective amount of a methylating protein intothe cell.

In one non-limiting embodiment, a lysine methyltransferase according tothe present invention, for example, vSET or a recombinantly producedvSET, can target a specific gene. In one non-limiting example, vSET canbe fused to a DNA binding protein domain that recognizes specificallythe promoter sequence of a given target gene, or to a histone bindingprotein domain that interacts with a core histone H3 or H4 carrying adistinct post-translational amino acid modification at the target genesite. In further non-limiting embodiments, vSET can be engineered topossess regulatory capacity via mutagenesis of amino acid residues atthe enzyme active site or methyl donor (S-adenosyl-methionine) co-factorbinding site, in which a small-molecule chemical compound can bedeveloped to control the enzymatic activity of vSET in a spatial andtemporal manner.

In Vivo Control of Gene Expression:

The present invention provides for methods of controlling the in vivoexpression of a gene in an organism or subject, for example, to treat acondition associated with increased gene expression, or a condition thatwould benefit by a decrease in transcriptional expression of one or moregenes, by administering to a subject in need of such treatment a lysinemethyltransferase, more preferably, a viral histone lysinemethyltransferase, more preferably, a protein comprising a Chlorellavirus SET domain of a viral histone lysine methyltransferase, and mostpreferably, a vSET or vSET-like viral histone lysine methyltransferase.“Decreasing or inhibiting the transcriptional expression of a targetgene in the subject” encompasses decreasing the level of transcriptionof a target gene in at least some, but not necessarily all cells,tissues, and/or fluids of the subject. The subject to be treated can bea subject who does not exhibit a mutation in the gene targeted fortranscriptional expression inhibition, but who would benefit fromdecreased transcriptional expression of the gene. The subject to betreated can also have a mutation in the gene targeted fortranscriptional expression inhibition, wherein the subject exhibitsincreased transcription and protein levels relative to cells thatnormally express the wild-type gene product of that gene. In oneembodiment, the subject is homozygous for the wild-type gene targetedfor transcriptional expression suppression. In another embodiment, thesubject is heterozygous for the wild-type gene and has a mutantgenotype, for example, a null genotype, for the other allele of the genetargeted for transcriptional expression inhibition.

In Vitro Control of Host Cell Gene Expression:

In another embodiment, the present invention provides for methods ofcontrolling the in vitro expression of a gene in a host cell, forexample, by inhibiting cell proliferation and/or promoting apoptosis ofa cell population, comprising administering to the cell population, alysine methyltransferase which decreases the transcriptional expressionof a target gene in the host cell. Inhibiting cell proliferation and/orpromoting apoptosis means decreasing the number of cells in a populationover a time interval, relative to a control population in which thetranscriptional expression of a target gene has not been decreased.

The term “host cell” means any cell of any organism that is selected,modified, transformed, grown, or used or manipulated in any way, for theproduction of a substance by the cell, for example the expression by thecell of a gene, a DNA or RNA sequence, a protein or an enzyme. In onenon-limiting embodiment, the host cell includes plant or mammalian hostcells. Suitable cells include, but are not limited to, PC12 cells, CHOcells, HeLa cells, HEK-293 (also known as 293 cells) and 293T (humankidney cells), COS cells (e.g. COS-7 cells), mouse primary myoblasts,NIH 3T3 cells.

In a preferred embodiment, the host cell is a cell of a eukaryotic cellline, for example, but not limited to HeLa cell lines; breast celllines; breast cancer cell lines such as MCF7 and MCF10A cell lines;prostate cancer cell lines such as PC3 and RWPE cell lines; leukemiccell lines such as K562, HL-60 and U937 cell lines; HEK-293T and COScells.

Cancer Treatment:

In related embodiments, the present invention provides for methods oftreating cancer, for example, by inhibiting tumor growth in a subject byinhibiting the expression of an oncogenic or tumorigenic gene, whichcomprises administering to the subject an effective amount of a lysinemethyltransferase which decreases the level of transcriptionalexpression of a target gene in the subject. Examples of malignancieswhich may be treated according to the present invention include, but arenot limited to, melanoma, glioblastoma multiforme, neuroblastoma,astrocytoma, osteosarcoma, breast cancer, cervical cancer, colon cancer,lung cancer, pancreatic cancer, Kaposi's sarcoma, hairy cell leukemia,nasopharynx cancer, ovarian cancer, and prostate cancer.

Treatment of Neurological Disorders:

The present invention also provides for methods of treating conditionssuch as degenerative disorders of the central nervous system (CNS), andperipheral nervous system (PNS), wherein the selective inhibition of atargeted gene or genes would be beneficial to a subject in need oftreatment. For example, a disease that is associated with neuronal orglial cell defects including, but not limited to, neuronal loss,neuronal degeneration, neuronal demyelination, gliosis (i.e.,astrogliosis), or neuronal or extraneuronal accumulation of aberrantproteins or toxins (e.g., β-amyloid, or α-synuclein). The neurologicaldisorder can be chronic or acute. Exemplary neurological disordersinclude, but are not limited to, Gaucher's disease, Parkinson's disease,Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiplesclerosis (MS), Huntington's disease, Fredrich's ataxia, Mild CognitiveImpairment, Cerebral Amyloid Angiopathy, Parkinsonism Disease, Lewy BodyDisease, Multiple System Atrophy (MSA), Progressive Supranuclear Palsy,and movement disorders (including ataxia, cerebral palsy,choreoathetosis, dystonia, Tourette's syndrome, kernicterus) and tremordisorders, and leukodystrophies (including adrenoleukodystrophy,metachromatic leukodystrophy, Canavan disease, Alexander disease,Pelizaeus-Merzbacher disease), neuronal ceroid lipofucsinoses, ataxiatelangectasia and Rett Syndrome.

The present invention is not limited in scope to the treatment of aparticular disease, or to the inhibition of a particular gene or classof genes associated with a disease state. The invention contemplates thetreatment of any disease or disorder characterized by an increase ingene expression as compared to a non-disease state, or a condition thatwould benefit from the inhibition of one or more genes' expression. Suchconditions may also include, but are not limited to, inflammatorydisorders, auto-immune disorders, or arthritic disorders.

Molecular Biology Definitions

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. These techniques are generallyuseful for the production of recombinant cells expressing lysinemethyltransferase proteins. Such techniques are explained fully in theliterature. (See, e.g., Sambrook, Fritsch & Maniatis, 2001, MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Glover, ed., 1985, DNACloning: A Practical Approach, Volumes I and II, Second Edition; Gait,M. J., ed., 1984, Oligonucleotide Synthesis: A practical approach;Hames, B. D. & Higgins, S. J. eds., 1985, Nucleic Acid Hybridization;Hames, B. D. & Higgins, S. J., eds., 1984, Transcription AndTranslation; Freshney, R. I., 2000, Culture of Animal Cells: A Manual ofBasic Technique; Woodward, J., 1986, Immobilized Cells And Enzymes: Apractical approach, IRL Press; Perbal, B. E., 1984, A Practical Guide ToMolecular Cloning).

Recombinant Lysine Methyltransferase

The lysine methyltransferase useful for the methods of the presentinvention can be isolated and purified using molecular biology,microbiology, and recombinant DNA techniques known to those of ordinaryskill in the art. For example, nucleic acids encoding the viral lysinemethyltransferase can be isolated using recombinant DNA techniques knownin the art. The nucleic acid encoding the lysine methyltransferase maybe full-length or truncated, as long as the gene encodes a biologicallyactive protein.

The identified and isolated gene encoding the viral lysinemethyltransferase can then be inserted into an appropriate cloningvector. A large number of vector-host systems known in the art may beused. Possible vectors include, but are not limited to, plasmids ormodified viruses, but the vector system must be compatible with the hostcell used. Examples of vectors include, but are not limited to, E. coli,bacteriophages such as lambda derivatives, or plasmids such as pBR322derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c,pFLAG, etc. The insertion into a cloning vector can, for example, beaccomplished by ligating the DNA fragment into a cloning vector whichhas complementary cohesive termini. However, if the complementaryrestriction sites used to fragment the DNA are not present in thecloning vector, the ends of the DNA molecules may be enzymaticallymodified. Alternatively, any site desired may be produced by ligatingnucleotide sequences (linkers) onto the DNA termini; these ligatedlinkers may comprise specific chemically synthesized oligonucleotidesencoding restriction endonuclease recognition sequences. Production ofthe recombinant protein can be maximized by genetic manipulations suchas including a signal peptide at the N terminus to facilitate secretionor a 3′ untranslated sequence containing a polyadenylation site.

In a preferred embodiment, the constructs used to transduce host cellsare viral-derived vectors, including but not limited to adenoviruses,adeno-associated viruses, lentivirus, herpes virus, mumps virus,poliovirus, retroviruses, Sindbis virus and vaccinia viruses.

Recombinant molecules can be introduced into host cells viatransformation, transfection, infection, electroporation, etc., so thatmany copies of the gene sequence are generated. Preferably, the clonedgene is contained on a shuttle vector plasmid, which provides forexpansion in a cloning cell, e.g., E. coli, and facile purification forsubsequent insertion into an appropriate expression cell line, if suchis desired.

Potential host-vector systems include, but are not limited to,eukaryotic cell systems, for example, mammalian cell systems infectedwith virus (e.g., vaccinia virus, adenovirus, lentivirus, etc.); insectcell systems infected with virus (e.g., baculovirus); microorganismssuch as yeast containing yeast vectors; or bacteria transformed withbacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elementsof vectors vary in their strengths and specificities. Depending on thehost-vector system utilized, any one of a number of suitabletranscription and translation elements may be used. Different host cellshave characteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g., glycosylation,cleavage [e.g., of signal sequence]) of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign protein expressed, such as glycosylation,sialyation and phosphorylation. For example, expression in a bacterialsystem can be used to produce a nonglycosylated core protein product.Expression in eukaryotic cells can increase the likelihood of “native”protein. Moreover, expression in mammalian cells can provide a tool forreconstituting, or constituting, protein. Furthermore, differentvector/host expression systems may affect processing reactions, such asproteolytic cleavages, to a different extent.

Purification of a recombinantly expressed protein can be achieved usingmethods known in the art such as by ammonium sulfate precipitation,column chromatography containing hydrophobic interaction resins, cationexchange resins, anion exchange resins, and chromatofocusing resins.Alternatively, immunoaffinity chromatography can be used to purify therecombinant protein using an appropriate polyclonal or monoclonalantibody that binds specifically to the protein, or to a tag that isfused to the recombinant protein. In a preferred embodiment, the purityof the recombinant protein used for the method of the present inventionwith be at least 95%, preferably 97% and most preferably, greater than98%.

Formulations

A lysine methyltransferase, more preferably a viral histone lysinemethyltransferase, more preferably a protein comprising a Chlorellavirus SET domain of a viral histone lysine methyltransferase, and mostpreferably a vSET or vSET-like histone lysine methyltransferase, asdescribed above, is advantageously formulated in a pharmaceuticalcomposition together with a pharmaceutically acceptable carrier. Thelysine methyltransferase may be designated as an active ingredient ortherapeutic agent for the treatment of a disease or disorder that wouldbenefit from a decrease in transcriptional expression of one or moregenes.

The concentration of the active ingredient (lysine methyltransferase,more preferably a viral histone lysine methyltransferase, morepreferably a protein comprising a Chlorella virus SET domain of a viralhistone lysine methyltransferase, and most preferably a vSET orvSET-like histone lysine methyltransferase) depends on the desireddosage and administration regimen. An artisan of ordinary skill candetermine the appropriate dosage using techniques that are routine inthe art.

In one embodiment, the lysine methyltransferase may comprise apharmaceutical formulation that is preferably suitable for parenteraladministration, including intravenous, subcutaneous, intra-arteriolar,intramuscular, intradermal, intraventricular, intrathecal, intracranialand intraperitoneal injection, however, formulations suitable for otherroutes of administration such as oral, intranasal, or transdermal arealso contemplated.

The pharmaceutical formulations suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. The preventions of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, benzyl alcohol, sorbic acid,and the like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonosterate and gelatin.

Sterile injectable solutions are prepared by incorporating the purifiedlysine methyltransferase in the required amount in the appropriatesolvent with various of the other ingredients enumerated above, asrequired, followed by filter or terminal sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andthe freeze-drying technique which yield a powder of the activeingredient plus any additional desired ingredient from previouslysterile-filtered solution thereof.

Preferably the formulation contains an excipient. Pharmaceuticallyacceptable excipients which may be included in the formulation arebuffers such as citrate buffer, phosphate buffer, acetate buffer, andbicarbonate buffer, amino acids, urea, alcohols, ascorbic acid,phospholipids; proteins, such as serum albumin, collagen, and gelatin;salts such as EDTA or EGTA, and sodium chloride; liposomes;polyvinylpyrollidone; sugars, such as dextran, mannitol, sorbitol, andglycerol; propylene glycol and polyethylene glycol (e.g., PEG-4000,PEG-6000); glycerol; glycine or other amino acids; and lipids. Buffersystems for use with the formulations include citrate; acetate;bicarbonate; and phosphate buffers. Phosphate buffer is a preferredembodiment.

The formulation also preferably contains a non-ionic detergent.Preferred non-ionic detergents include Polysorbate 20, Polysorbate 80,Triton X-100, Triton X-114, Nonidet P-40, Octyl .alpha.-glucoside, Octyl.beta.-glucoside, Brij 35, Pluronic, and Tween 20.

The lysine methyltransferase formulation may be subject tolyophilization. Bulking agents, such as glycine, mannitol, albumin, anddextran, can be added to the lyophilization mixture. In addition,possible cryoprotectants, such as disaccharides, amino acids, and PEG,can be added to the lyophilization mixture. Any of the buffers,excipients, and detergents listed above, can also be added.

Formulations for inhalation administration may contain lactose or otherexcipients, or may be aqueous solutions which may containpolyoxyethylene-9-lauryl ether, glycocholate or deoxycocholate. Apreferred inhalation aerosol is characterized by having particles ofsmall mass density and large size. Particles with mass densities lessthan 0.4 gram per cubic centimeter and mean diameters exceeding 5 μmefficiently deliver inhaled therapeutics into the systemic circulation.Such particles are inspired deep into the lungs and escape the lungs'natural clearance mechanisms until the inhaled particles deliver theirtherapeutic payload. (Edwards et al., Science 1997; 276: 1868-1872).Protein preparations of the present invention can be administered inaerosolized form, for example by using methods of preparation andformulations as described in, U.S. Pat. Nos. 5,654,007, 5,780,014, and5,814,607, each incorporated herein by reference. Formulation forintranasal administration may include oily solutions for administrationin the form of nasal drops, or as a gel to be applied intranasally.

Formulations for topical administration to the skin surface may beprepared by dispersing the composition with a dermatological acceptablecarrier such as a lotion, cream, ointment, or soap. Particularly usefulare carriers capable of forming a film or layer over the skin tolocalize application and inhibit removal. For topical administration tointernal tissue surfaces, the composition may be dispersed in a liquidtissue adhesive or other substance known to enhance adsorption to atissue surface. Alternatively, tissue-coating solutions, such aspectin-containing formulations may be used.

In preferred embodiments, the formulations of the invention are suppliedin either liquid or powdered formulations in devices which convenientlyadminister a predetermined dose of the preparation; examples of suchdevices include a needle-less injector for either subcutaneous orintramuscular injection, and a metered aerosol delivery device. In otherinstances, the preparation may be supplied in a form suitable forsustained release, such as in a patch or dressing to be applied to theskin for transdermal administration, or via erodable devices fortransmucosal administration.

Administration of the above-described parenteral formulations may be byperiodic injections of a bolus of the preparation, or may beadministered by intravenous or intraperitoneal administration from areservoir which is external (e.g., an i.v. bag) or internal (e.g., abioerodable implant, a bioartificial organ, or a population of implantedcells that produce the replacement protein). See, e.g., U.S. Pat. Nos.4,407,957 and 5,798,113, each incorporated herein by reference.Intrapulmonary delivery methods and apparatus are described, forexample, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607, eachincorporated herein by reference. Other useful parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, pump delivery, encapsulated celldelivery, liposomal delivery, needle-delivered injection, needle-lessinjection, nebulizer, aeorosolizer, electroporation, and transdermalpatch. Needle-less injector devices are described in U.S. Pat. Nos.5,879,327; 5,520,639; 5,846,233 and 5,704,911, the specifications ofwhich are herein incorporated by reference. Any of the formulationsdescribed above can administered in these methods.

In a specific embodiment, the protein or vector therapeutic compositionis delivered directly to the target tissue, e.g., a tumor, particularlyfor an unmodified lysine methyltranferase therapeutic product. Thisensures that the effect is greatest where it is needed, and limitssystemic side effects that might be undesirable.

EXAMPLES

The present invention is further described by means of the examples,presented below. The use of such examples are illustrative only and inno way limits the scope and meaning of the invention or of anyexemplified term. Likewise, the invention is not limited to anyparticular preferred embodiments described herein. Indeed, manymodifications and variations of the invention will be apparent to thoseskilled in the art upon reading this specification. The invention istherefore to be limited only by the terms of the appended claims alongwith the full scope of equivalents to which the claims are entitled.

Example 1 vSET and vSET-Like Proteins from Chlorella Viruses SuppressGene Transcription Through Methylation of H3-K27

Methods

Growth of Virus and Preparation of Chlorella Cell Extracts

Growth of Chlorella NC64A on MBBM medium, the plaque assay, theproduction of the viruses, and the isolation of virus DNAs were carriedout as described previously (Schuster, A. M. et al. Characterization ofviruses infecting a eukaryotic chlorella-like green alga. Virology 150,170-7 (1986)). Actively growing Chlorella NC64A cells (3×10¹⁰ cells in150 ml) were mock infected or infected with virus PBCV-1 at amultiplicity of infection of 5 for various times. Purified PBCV-1 virusparticles (200 μl of 6 mg/ml total virus) were treated with 200 U/mlsequencing grade modified trypsin (Promega) prior to furtherpurification. This treatment has not effect on virus infectivity.

RNA Level Analysis of Infected Chlorella

Actively growing Chlorella NC64A cells (1-2×10⁷ per ml) were incubatedwith 5 μl/ml [³H]-adenine (37 MBq/ml) for 10 minutes, centrifuged andcells were re-suspended in 50 mM Tris-HCl, pH 7.8, (5×10⁷ cells per ml)plus PBCV-1 at a multiplicity of infection of 10. One ml samples werecollected at appropriate times and mixed with an equal volume of icecold 10% trichloroacetic acid (TCA). Cells were collected on glassfilters, washed three times with 5% TCA, one time with 80% ethanol anddried. RNA was hydrolyzed by placing the filters in 5 ml of 0.5 M NaOHovernight at 37° C., collected by filtering through a fresh filter,neutralized with TCA, and analyzed by scintillation counting. Total RNAwas isolated from PBCV-1 infected Chlorella cells at various times postviral infection using the Trizol reagent (Invitrogen). Total RNA waselectrophoresed and hybridized with a ³²P-labeled gene probe using aRandom-Primer DNA Labeling Kit (Invitrogen). Viral DNAs used for dotblots were denatured, applied to nylon membranes, fixed by UVcross-linking, and hybridized with a gene probe used for the Northernanalysis.

DNA Cloning, Protein Purification, and Histone Methyltransferase Assay

Full-length histone H3 DNA from Chlorella NC64A was cloned by designingdegenerate primers based on H3 from Chlamydomonas reindardtii. The SETgene was isolated from chlorella viruses MA-1D, NY-2A and NY-2B using anestablished protoco. (Kang, M. et al. Genetic diversity in chlorellaviruses flanking kcv, a gene that encodes a potassium ion channelprotein. Virology 326, 150-9 (2004)) The DNA bands that hybridizedweakly with the PBCV-1 vSET probe were excised from the gel, recoveredwith a QIAEX II Gel Extraction Kit (Qiagen) and cloned into pGEM-7Zf(+).Primers were designed according to the vSET homologs identified fromthese hybridized DNA fragments. The vSET homologs from these threeviruses were amplified by PCR and subcloned into pET-15b (Novagen). AllDNA constructs and vSET mutants prepared using the QuikChangemutagenesis kit (Stratagene) were confirmed by DNA sequencing.Recombinant proteins were expressed in E. coli strain BL21. The in vitrohistone methyltransferase reaction was performed by following methyltransfer from S-adenosyl-[¹⁴C-methyl]-L-methionine (Amersham) to histonepeptide or nucleosome substrates, or by mass spectroscopy analysis ofthe histone peptide substrates (Qian, C. et al. Structural insights ofthe specificity and catalysis of a viral histone H3 lysine 27methyltransferase. J Mol Biol 359, 86-96 (2006)). Upstream anddownstream primer sequences used for histine H3 are 5′-ATGGCCCGCACCAAG(SEQ ID NO:8) and 5′-TTAGGCGCGCTCGCC (SEQ ID NO:9), respectively.

Western Blot and Histone Immunoprecipitation Analyses

PBCV-1 infected Chlorella cells were harvested at different times postinfection. Western blot analysis of the immunoprecipitated protein wasdone using anti-vSET antibodies, generated in rabbits againstrecombinant vSET (Covance). Histone immuno-precipitation was performedon Chlorella cells collected at 0-300 minutes post PBCV-1 infection. Theassay was carried out by using anti-histone H3, H3-K27me1, H3-K27me2,H3-K27me3, H3-K27ac and H3-S28p antibodies (Millipore) with cellscross-linked with paraform-aldehyde and sheared by sonication. To assessthe PcG proteins at H3-K27, HeLa cells stably transfected with vSET andtreated with or without tetracycline, were subject to crosslinking andimmunoprectipation using antibodies against EZH2 and Polycomb complexproteins, RING1, SUZ12, Ezh2/PRC2, Bmi/PRC1, CBX4, CBX7 and CBX8, andfollowed by Western blots using anti-H3 antibodies.

Nuclear Localization and Co-Localization

vSET nuclear localization was analyzed in NIH-3T3 cells transientlytransfected with a pEGFP-N1 vector (Clontech) that encodes a C-terminalGFP fusion vSET. The analysis includes fixation, permeablization andimmunofluorescence. Typically, the exponentially growing cells werecollected and suspended in 2% paraformaldehyde in PBS buffer of pH 7.4at 4° C. for 6 hours. The fixed cells were pelleted by centrifugation,re-suspended in 5 ml chilled methanol, washed with PBS, and spotted ontoa slide and air-dried. Cells adhered to the slides were incubated for 15minutes at 4° C. in DMSO (0.5% vol/vol in PBS). For fluorescenceanalysis, cells were blocked with 0.5% bovine serum album in PBS andincubated with anti-vSET and anti-histone antibodies (Abcam) for 1 hourat 25° C. Cells were washed with PBS, and incubated with secondaryfluorescein labeled antibody (Alexa 488), mounted with a cover slip andanalyzed using a Zeiss Axioplan2 microscope. For the co-localizationassay, staining was done with rabbit polyclonal anti-vSET and anti-ratmonoclonal H3-K27me2 antibodies. The respective immune complexes weredetected using Alexa488 and 594 (Molecular Probes).

Transcription Reporter Assay

A luciferase reporter gene transcription assay was performed asdescribed previously (Nishio & Walsh, Proc Natl Acad Sci USA 101,11257-62 (2004)). Briefly, 293T cells were co-transfected with 2 μg ofpcDNA3-Ga14-DBD-vSET (or the mutants) and 1 μg of HSV-tk-promoter plusGal4 binding sitre and luciferase gene constructs (e.g.,pGL2-Ga14-E1b-Luc constructs). A luciferase assay on HOX7A promoter wasalso performed upon co-transfection with Renilla luciferase in Helacells with and without EZH2 knockdown by siRNA, as described below. vSETrepression of Tat-mediated transcription of a HIV LTR-luciferase genewas also evaluated. For this study, vSET and HIV Tat were transfectedinto HeLa (TZM-bl) cells that contain an integrated HIV LTR-luciferasereporter gene, or 293T cells with a HIV LTR-luciferase gene and HIV Tatwith or without vSET (Derdeyn et al., J Virol 74, 8358-67 (2000);Mujtaba, S. et al., supra). After 48 hours, cells were lysed and assayedfor luciferase activity using the Bright-Glo luciferase assay system(Promega). Each assay was done in duplicate and repeated five times.Chromatin immunoprecipitation (ChIP) analysis was also performed usingEZ-ChIP kit (Millipore) to evaluate vSET repression of Tat-dependent HIVLTR-luciferase reporter gene transcription in Hela cells at the HIVpromoter located in the nucleosome Nuc2 by using various antibodies forhistone H3 of different modifications.

Cell Cycle Analysis

The vSET effect on the cell cycle was analyzed in NIH-3T3 cellstransiently transfected with vSET in a pCMV-tag2B vector (Stratagene)and a Us9-GFP encoding plasmid, or HeLa cells stably co-transfected withvSET in a tetracycline-controlled vector pcDNA4/TO (Invitrogen) and theUs9-GFP plasmid. Post-transfection cells were treated with 1 μg oftetracycline (1 μg ml⁻¹), harvested after 24 hours by trypsinization,washed with PBS and fixed in chilled 70% ethanol in PBS. One hour beforeacquiring the data, cells were washed again with PBS and stained withpropidium iodide (PI). Cell cycle analysis was performed with a Caliburflow cytometer (Becton Dickinson) after GFP and PI staining.

EZH2 RNAi Knockdown, CHIP, and Quantitative RT-PCR Analyses

RNAi knockdown of EZH2 was performed in Hela cells with target-specificand smart-pool siRNAs from Dharmacon. Cell transfection with siRNAs wasdone according to manufacturer's instructions. EZH2 RNAi knockdown wasevaluated 72 hours after transfection by Western blot analysis usinganti-EZH2 antibody (Cell Signaling). Methylation states of K4, K9, K27and K36 on histone H3 were evaluated by using various antibodiesobtained from Millpore and Abcam. Chromatin immunoprecipitation (ChIP)analysis was performed using EZ-ChIP kit (Millipore) to evaluate vSETrepression of Tat-mediated transcription of the HIV LTR-luciferasereporter gene in Hela cells on the HIV promoter sequence in Nuc2 usingvarious antibodies for histone H3 of different modifications. For RT-PCRanalysis, RNA was extracted using RNAeasy kit (Qiagen) from Hela cellsthat were transfected with EZH2 siRNA and/or vSET. The primers fortarget genes of choice for analysis were designed based on the publishedsequences (Svingen et al.). cDNA was generated by RT-PCR using theaffinity script from Stratagene. Reactions were determined usng the SYBRGreen I detection chemistry system (Applied Biosystems) with an ABIPrism 7300 Sequence Detection System. Chromatin immuno-precipitation(ChIP) analysis was performed for the HOXA7 gene using the EZ-ChIP kit(Millipore) following the manufacturer's instruction as previouslydescribed (De Santa et al., Cell 130:1083-1094 (2007)).

Dot Blot for Antibody Specificity

Dot blot assay was performed on 0.2 μm nitrocellulose paper (GEHealthcare; catalog #RPN3032D). Histone peptides (500 μM each) werespotted on the nitrocellulose paper and dried for 1 hour at roomtemperatures. Subsequently, the membrane was blocked for 1 hour at roomtemperature with 2% non-fat milk in TBS buffer. After blocking, themembrane was washed with TBS, and treated with respective antibodies for2 hours at room temperature. After antibody exposure, the membrane waswashed three times for 5 minutes each with TBS containing 0.05%Tween-20. Finally, the membrane was treated with secondary antibody (GEHealthcare; catalog #NA9340V) for 1 hour at room temperature. Afterwashing three times for 5 minutes each with the TBS Tween-20 buffer themembrane was exposed to ECL reagent (GE Healthcare; catalog #RPN2106)(Huang et al., Nature 449:105-108 (2007); Erhardt et al., Development130:4235-4248 (2003); and Rougeulle et al., Mol Cell Biol. 24:5475-5484(2004)).

Histine as the Major Target of vSET

Heat inactivated cell extracts from EZH2-knockdown HeLa and chlorellacells were subjected to vSET enzyme assay, and subsequently analyzedwith western blot by using anti-H3, H3K27me2, H3K9me2, H3K4me2, andPan-methylated lysine (Pan-Kme) antibodies. After antibody exposure, themembrane was washed three times for 5 minutes each with TBS buffercontaining 0.05% Tween-20. The membrane was treated with secondaryantibody (GE Healthcare; catalog #NA9340V) for one hour at roomtemperature. Finally, after washing three times for 5 minutes each withthe TBS buffer the membrane was exposed to ECL reagent (GE Healthcare;catalog #RPN2106).

Luciferase Reporter Gene Assay

Gene repression activity by vSET was measured in transient transfectionluciferase reporter gene assay in 293T cells. The constructs used in thestudy presented in FIG. 4a are Ga14-DBD (aa 1-147), Ga14-DBD-vSET,Ga14-DBD-vSET-Y105A and Y105F, Ga14-tk-luciferase and renillaluciferase, and in FIG. 4b are Ga14-DBD (1-147), Flag-vSET and FlagvSET-Y105F, and HAvSET and HA-vSET-Y105F. The results are presented asrelative luciferase expression in the presence of GA4-DBD-vSET ascompared to that with GAL4-DBD alone. The reporter luciferase gene islinked to a Ga14-tk-luciferase promoter construct. Luciferase activitylevels were determined using the dual luciferase kit (Promega) and datawere normalized to the activity of a co-transfected renilla luciferaseplasmid (Promega). HOXA7 luciferase was performed by co-transfectingHeLa cells with HOXA7 promoter and renilla luciferase in the presence orabsence of EZH2. HOXA7 promoter was a gift from Drs. R. Slaney and K.Kamps. Both GAL4 and HOXA7 luciferase assays were performed on threedifferent days. The error bars represent the standard deviation ofluciferase levels measured in three different data sets.

Results

vSET is a Structural Protein of PBCV-1 Virions

Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virusthat replicates in the unicellular, eukaryotic, green alga Chlorellastrain NC64A; Chlorella species are one of the most widely distributedand frequently encountered groups of algae on earth (Van Etten, Annu RevGenet 37, 153-95 (2003)). The 330-kb PBCV-1 genome has 366non-overlapping protein-encoding genes and 11 tRNA genes that have amosaic of prokaryotic- and eukaryotic-like proteins (Van Etten, 2003.).Despite its large genome, the virus lacks a recognizable RNA polymerasegene, suggesting that it is dependent on host enzyme(s) fortranscription. Notably, PBCV-1 infection results in a rapid decrease intotal RNA synthesis reaching 60-80% inhibition 20-40 minutes postinfection (FIG. 1a ). Rapid transition from host to virus transcriptionoccurs as PBCV-1 transcripts can be detected 5-10 minutes post infection(Van Etten, 2003). These results lead to the postulation that afactor(s) packaged in the virion might be responsible for the rapidinhibition of host RNA synthesis. vSET is a good candidate for such afactor, because vSET methylates H3-K27 (see below), which has beenlinked in eukaryotes to the PcG complex-mediated Hox gene silencing(Czermin et al., Muller, J. et al., Cao et al., Kuzmichev et al., Plath,K. et al., Boggs, B. A. et al., Bernstein, B. E. et al., Boyer, L. A. etal., Lee, T. I. et al. Cao & Zhang; supra).

To investigate the role of vSET in PBCV-1 infection, an anti-vSETantibody was produced and used to immunoprecipitate vSET from viralinfected Chlorella cell extracts. As shown in FIG. 1b , vSET is presentin mature virions and a small amount is detected in virus infectedChlorella cells as early as 10 minutes post infection; vSET appears inlarge quantities by 120 minutes after virus infection. Trypsin treatmentof intact PBCV-1 particles eliminated the possibility that vSET is acontaminant on the virion surface (FIG. 1c ). The presence of vSET inPBCV-1 virion particles was also confirmed by Q-TOF mass spectrometry(Dunigan, Cerny and Van Etten, unpublished results). Comparing westernblots from a known number of virus particles with blots containingdifferent concentrations of vSET led to abn estimation that four vSETmolecules are packaged per virion (data not shown). Furthermore, vSET inthe disrupted virions shows histone H3-K27 methylation activity (FIG. 1e). Therefore functional vSET is packaged in the virions.

To determine when vset is transcribed during PBCV-1 replication, RNAfrom infected Chlorella cells was probed with the vset gene. The probehybridized to an RNA of ˜1.8 kb beginning at ˜60 minutes post infection(FIG. 1d ). This transcript is larger than expected for a 119-residueprotein and may be a bicistronic transcript encoding the co-linear genesa609l (˜1.2 kb) and a6121 (˜0.4 kb). The latter gene encodes vSET. ThisNorthern result is consistent with vSET expression occurring ˜120minutes after virus infection (FIG. 1b ). The observation that vset isexpressed as a late gene agrees with the finding that vSET is present inmature PBCV-1 virions because virion associated proteins are usuallylate gene products (Van Etten, 2003).

PBCV-1 vSET is a Bona Fide HKMTase

vSET adopts a core beta-barrel structure, a fold that is conserved ineukaryotic SET domain HKMTases (Manzur et al., Qian et al., Qian & Zhou,supra). To test whether vSET is a bona fide HKMTase of the hostchlorella, histone H3 from Chlorella NC64A was cloned and sequenced. TheChlorella H3 has high sequence identity to H3 from human, mouse,Caenorhabditis elegans as well as the green alga Chlamydomonasreinhardtii (FIG. 2a ). vSET methylation activity was measured usingboth the nucleosome and individual core histones, confirming itsactivity for full-length free H3 and H3 within the nucleosome, but notother core histones (FIG. 2b ). To identify vSET methylation site(s) inH3, a series of GST-H3 peptides were prepared (residues 1-57) witharginine substitutions at lysine methylation sites, K4, K9, K27, K36 andK37. The purified GST-H3 peptides produced two bands whenelectrophoresed on SDS-PAGE that corresponded to an intact H3 peptide ofresidues 1-57 (plus SGRIVTD (SEQ ID NO:53) from the expression vector)and a truncated H3 of residues 1-55, as confirmed by MALDI-TOF massspectrometry (FIG. 2c ). The methylation assay showed that vSET was onlyactive when H3 contained K27 (FIG. 2c ). Using antibodies against site-and state-specific lysine methylated histone H3, it was confirmed thatvSET can predominately catalyze di-methylation at H3-K27 and to a muchless extent mono- and tri-methylation, and not methylation at K4, K9 orK36 in H3 (FIG. 2d ). The former is consistent with the enzyme kineticsanalysis of vSET methylation of H3-K27 peptide, showing thatmono-methylation and mono- to di-methylation are very rapid whereas di-to tr-methylation is ˜10 times slower than mono- to di-methylation(FIGS. 2H and 2I). Collectively, these results clearly show that vSET isH3-K27 specific di-methylase.

Possible changes in H3-K27 in Chlorella NC64A cells after PBCV-1infection were examined. As revealed by Western blot analysis usingantibodies specific for histone H3 with different modifications (FIG.6a-e ), host H3-K27me1 was largely unchanged after viral infection,whereas H3-K27me2 was increased markedly as early as 30 minutes afterviral infection, and H3-K27me3 was enhanced slightly (FIG. 2e ). Thisobservation correlates well with in vitro vSET state-specificmethylation activity at H3-K27 (FIG. 2d ). As illustrated byimmunofluorescence, vSET was present in the nucleus of the hostChlorella cells after PBCV-1 infection (FIG. 2f ), and vSET co-localizedwith the host H3 that became di-methylated at K27 (FIG. 2g ).

vSET Contains a Nuclear Localization Signal

Due to the technical difficulties of manipulating the PBCV-1 genome andgenetic transformation of the host chlorella cells, it is not possiblecurrently to conduct a cellular study of vSET in its native host.However, because of the highly conserved H3 sequence and H3-K27methylation in eukaryotes, it was reasoned that mammalian cells couldserve as a suitable model system to study the biological function ofvSET, which as similar enzymatic activity as EZH2 (Cao et al, supra.).

For vSET to participate in early suppression of host transcription, itmust move to the host nucleus immediately after viral infection. Toassess whether a solvent-exposed KRMR (SEQ ID NO:3) motif in vSET(residues 85-88, FIG. 5b ) functions as a nuclear localization signal(NLS), NIH-3T3 cells were transfected with either a GFP-fusion vSET or atriple mutant where KR(M)R (SEQ ID NO:3) was changed to AA(M)A(SEQ IDNO:4). Immunofluorescence showed that wild-type GFP-vSET was localizedto the nucleus, whereas the triple mutant was in the cytoplasm (FIG. 3a), confirming that the KR(M)R (SEQ ID NO:3) motif is an NLS in vSET. Itwas established that H3-K27 methylation activity and vSET proteinconformation was not compromised in this mutant (FIG. 7a-c ). Note thata classical NLS sequence is absent in most eukaryotic SET domains,except for yeast SET1 that has a RRIV (SEQ ID NO:54) motif located at asite analogous to the KRMR (SEQ ID NO:3) motif in vSET. Viral proteinssuch as human adenovirus-5 E1A protein and bovine herpesvirus-1 VP8protein contain basic KRPR (SEQ ID NO:6) and RRPR (SEQ ID NO:7) patches,respectively, indicating that putative NLS signals are encoded in virusproteins (Douglas, J. L. & Quinlan, M. P. Structural limitations of theAd5 E1A 12S nuclear localization signal. Virology 220, 339-49 (1996);Zheng et al., Characterization of nuclear localization and exportsignals of the major tegument protein VP8 of bovine herpesvirus-1.Virology 324, 327-39 (2004)).

vSET Mimics Mammalian EZH2 for H3-K27 Methylation

To investigate whether vSET can methylate H3-K27 in mammalian cells,RNAi knockdown of EZH2 was performed. As shown in FIG. 3b , EZH2expression was reduced by greater than 90% in Hela cells after treatedwith EZH2-specific siRNA as compared to control. EZH2 knockdown alsoresulted in a marked reduction of SUZ12, a component of the PRC1complex, as well as a nearly complete loss of H3-K27me2 and K27me3 and alittle effect on H3-K27me1. No significant change was observed withH3-K4, H3-K9 and H3-K36 di-methylation. These results confirm that EZH2is responsible for H3-K27 di- and tri-methylation in Hela cells. vSETtreatment of the nuclear extract of the EZH2 knocked-down Hela cellsrestore H3-K27me2 and K27me3, and results in a slight reduction ofH3-K27me1, possibly due to its conversion to H3-K27me2 and K27me3 byvSET (FIG. 3c ). There was no change in the level of H3-K4, K9 and K36methylation. These results are similar to effects oftetracycline-induced vSET expression in the EZH2 knocked-down Hela cells(FIG. 3d ). Taken together, the results strongly demonstrate that vSETcan mimic EZH2 of the PRC2 complex for methylating H3-K27 in mammaliancells.

vSET Induces Gene Silencing

The effect of vSET methylation activity on host transcription wasexamined using a luciferase reporter assay in human embryonic kidney293T cells. The study was first carried out with the 293T cellstransfected with vSET fused to a Gal4 DNA-binding domain (DBD) and aluciferase reporter gene encoding HSV-tk promoter (plus Gal4 DNA-bindingsites). As shown in FIG. 4a , wild-type vSET caused ˜95% repression ofthe luciferase gene expression, whereas its inactive mutant Y105A orY105F completely lost vSET gene silencing ability. Moreover, expressionof a Flag- or HA-tagged vSET in the 293T cells exhibited ˜60% repressionas compared to the vectors containing only Flag- or HA-tag, or inactivemutant Y105F (FIG. 4a ). The less profound Flag- or HA-tagged vSETrepressive activity (a global effect) on host transcription as comparedto that of vSET fused directly to Ga14-DBD (a targeted effect) may be inpart due to the existence of H3-K27 acetylation and/or H3-S28phosphorylation in host cells that would preclude H3-K27 methylation byvSET. Finally, it was observed that expression of a Flag-tagged vSETco-transfected with HIV Tat in 293T cells (FIG. 6d ) or HeLa cells (FIG.6e ) causes transcription repression of a Tat-mediated HIVLTR-luciferase gene in a dose-dependent manner, thus confirming thatvSET methylation activity represses chromatin-mediated genetranscription. (Mujtaba, S. et al., Dorr, A. et al., supra).

vSET Promotes PRC1 CBX8 Recruitment to 113-K27me Site

Histone H3-K27 methylation is catalyzed by the EZH2/PRC2 complex andrecruits the PRC1 complex in eukaryotes leading to gene silencing (Caoand Zhang; Czermin et al.; Muller et al.; Cao et al.; Kuzmichev et al.).To determine whether vSET gene transcription repression is due to itsH3-K27 methylation activity, Tat-mediated HIV LTR-luciferase activationand chromatin immunoprecipitation (ChIP) analyses were performed on theHIV LTR promoter in Hela TZM cells transfected with Flag-tagged Tat andvSET. As shown in FIG. 4c , Tat-mediated LTR activation was reduced bymore than 90% upon expression of vSET but not its inactive mutant Y105F.An enhanced acetylation on H3 was observed at Nuc2 where the LTRpromoter is localized but not at housekeeping gene GAPDH site when cellsare transfected only with HIV Tat. These effects by vSET coincide with amarked increase of H3-K27me2 and to a lesser extent H3-K27me3 (FIG. 4d). Conversely, H3-K27me1 was somewhat decreased upon vSET expression,which is consistent with the observation of vSET treatment of the EZH2knocked-down Hela cells (FIG. 3b-d ).

To determine the biological consequence of H3-K27 methylation by vSET,Polycomb group protein occupation at the H3-K27 site was examined upontetracycline-induction of vSET in the EZH2 knocked-down Hela cells. Itwas observed that while their protein levels are not altered, vSETinduction enhanced occupation of the PRC1 complex protein CBX8, but notCBX4 and CBX7, or the PRC2 RING2 at H3-K27me2 and H3-K27me3 sites (FIG.4e ). This methylation-dependent CBX8/H3-K27me2 association is likelyfacilitated by the methyl-lysine binding chromodomain of CBX8 (Min etal., Genes Dev 17:1823-1828 (2003)). This vSET-induced CBX8/H3-K27me2association was also seen in ChIP on Nuc2 of the HIV LTR promoter (FIG.4d ), as well as in the regular Hela cells transfected with vSET (FIG.6c ). Taken together, these results confirm that vSET induces genesilencing via its H3-K27 methylation activity, and facilitates theconsequent PRC1 complex CBX8 recruitment to chromatin site of H3-K27methylation enhanced by vSET.

vSET Modulates Polycomb Target Genes

Whether vSET methylation activity can modulate Polycomb target genes wasdetermined using a quantitative RT-PCR assay (Bracken et al.). Theanalysis was performed in Hela cells on five representative Polycombtarget genes HOXA7, HOXA9, HOXB9, HOXD8, and Hey1; and three generalhousekeeping genes GAPDH, RPS, and tubulin. While the effect on thethree housekeeping genes was negligible, EZH2 knockdown by siRNAresulted in a ˜2.5-fold increase in transcription of HOXA9, HOXB9, HOXD8and Hey1, and a striking 7.5-fold increase in HOXA7 expression (FIG. 4f). The enhancement of the Polycomb target genes' expression was reversedupon tetracycline-induced expression of vSET to a level at or slightlyhigher than that of the control cells. Moreover, vSET induction in theregular Hela cells does not cause any significant change in theexpression level of all five HOX genes as compared to the controls.

vSET transcription repression was characterized on the HOXA7 promoter inluciferase gene expression and chromatin immunoprecipitation (ChIP)assays. Upon induction vSET effectively represses the luciferaseexpression at the HOXA7 promoter in both EZH2 siRNA treated and regularHela cells (FIG. 4i , and FIG. 6F). the ChIP analysis confirmed thatvSET specifically di- and tri-methylates H3-K27 at the HOXA7 promoter,which recruits CBX8 of the PRC1 resulting in gene repression (FIG. 4j ).Similar results were obtained in the characterization of vSETtranscription repression of Tat-dependent HIV LTR-luciferase reportergene at the nucleosome Nuc2 where the HIV LTR promoter is localized inthe Hela TZM cells transfected with Tat and vSET (see FIGS. 4c and 4d ).These results establish that vSET can modulate transcription of Polycombtarget genes through its H3-K27 methylation activity.

vSET Causes Cell Accumulation at G2/M Phase

The effect of vSET expression on the normal cell cycle was examined.Flow cytometric analysis indicated that vSET caused cell accumulation atthe G2/M phase in transiently transfected NIH-3T3 cells, whereasactive-site mutants E100A and Y105A had less or nearly no effect (FIG.4g ). The vSET effect on G2/M cell cycle arrest was confirmed in thestudy using the HeLa cells that were stably co-transfected with vSET ina tetracycline-controlled pcDNA4/TO expression vector and an Us9-GFPencoding plasmid (FIG. 4h ). Collectively, the data indicate that vSETH3-K27 methylation activity results in the recruitment of the Polycombgroup protein complexes and modulation of their target genes, leading totranscription repression and accumulation of cells at the G2/M phase.

vSET-Like Proteins are Universally Encoded by Chlorella Viruses

To determine if vSET is functionally unique or universal in thechlorella viruses, genomic DNA from 36 other chlorella viruses washybridized with a vset gene probe. Thirty-one of these viruseshybridized to the probe (FIG. 5a ). The probe did not hybridize to DNAfrom 5 viruses or the host DNA. However, a vset gene was identified in 3of these 5 viruses using low stringency Southern hybridization. The vSETproteins from these three viruses, NY-2A, NY-2B and MA-1D, have 85%amino acid identity to PBCV-1 vSET, including the conserved active-siteresidues in the SET domain family (FIG. 5b ).

Using core histones H2A, H2B, H3 and H4 as substrates, it wasestablished that the SET proteins from these 3 viruses selectivelymethylate H3 (data not shown). Like vSET, these viral SET proteins onlymethylated a H3 peptide containing residues 15-30 but not a peptidecontaining H3 residues 1-20 (FIG. 5c ). They showed littlemethyltransferase activity if the H3 peptide was previouslydi-methylated at K27 or phosphorylated at S28, suggesting a preferredmethylation site at H3-K27. The selective methylation at H3-K27 wasconfirmed using GST-histone H3 (residues 1-57), in which these viral SETproteins had robust methylation activity on wild-type H3 but little ordiminished activity on the K27R mutant (FIG. 5d ). Collectively, theseresults establish that SET proteins from the chlorella viruses possessselective H3-K27 methylation activity, and suggest that vSET function inhost chromatin modification and transcription repression is conservedamong the chlorella viruses.

Discussion

Histone modifications provide epigenetic control of gene transcriptionon the chromatin. Viruses recruit host cellular proteins to eitherintegrate their genomes into host chromatin or to facilitate viraltranscription and replication (You et al., Wu et al., Ghosh et al., Donet al., supra). Here, it has been shown for the first time that virusestake control of the host transcription machinery via directmodifications of chromatin. This study reveals that vSET is packaged inthe PBCV-1 virion and is capable of directly methylating host H3-K27, animportant epigenetic modification in eukaryotes that has beenfunctionally linked to Hox gene silencing, X-inactivation and stem cellpluripotency (Czermin et al., Muller et al., Cao et al., Kuzmichev etal., Cao & Zhang, Plath et al., Boggs et al., Bernstein et al., Boyer etal., Lee et al., supra). The results establish that vSET H3-K27methylation promotes the recruitment of the PRC1 complex via a molecularinteraction between the chromodomain of CBX8 and di- and/ortri-methylated H3-K27 that results in transcriptional repression andcell accumulation at the G2/M phase in cell cycle in the continuouslydividing cells (Czermin et al., Muller et al., Cao et al., Kuzmichev etal., Cao & Zhang, Plath et al., Boggs et al., Bernstein et al., Boyer etal., Lee et al., supra). These results support a hypothesis that therole of vSET is to globally shut off host gene transcription in infectedchlorella cells. The demonstration that a protein with H3-K27methylation activity is encoded by all the chlorella viruses providesadditional support for this hypothesis.

Several unusual features of vSET emerged from this study and highlightits efficiency as a viral transcription suppressor. First, vSET ispackaged in mature virions, and becomes active immediately after viralinfection and host cell entry. Second, vSET has a nuclear localizationsignal that allows transport to the host nucleus. Third, vSET has highsubstrate specificity for H3-K27 but not H3-K9 sites even though the twosites have similar amino acid sequences (Qian et al., supra). Fourth,the literature on functional significance of H3-K27 mono-methylationremains elusive, and how H3-K27 di- and/or tri-methylation isfunctionally linked to development and gene silencing is also equivocal(Cao & Zhang, supra). vSET can be an important tool for resolvingquestions about the role of H3-K27 methylation states on cellularprocesses as the ability of vSET to produce mono-, di-, andtri-methylations at this site can be controlled by amino acidsubstitutions in vSET at its active site (Qian et al., supra). Finally,vSET is the smallest SET domain HKMTase; vSET lacks the pre- andpost-SET motifs present in mammalian SET HKMTases (Qian & Zhou, supra).In contrast to mammalian monomeric SET HKMTases, vSET functions as adimer. It may be postulated that in the quest for efficiency, thedimeric form of this viral SET HKMTase may have evolved to allowmethylation of histone pairs within one nucleosome or in neighboringnucleosomes, thereby increasing its efficiency in modifying hostchromatin.

The presence of a viral encoded and virion packaged enzyme with directchromatin-modifying properties may provide advantages to the chlorellaviruses. For example, it would eliminate the need for using cellularproteins as functional mediators and therefore, the expression of suchcellular proteins would not be a factor. It would also increase theefficiency with which the virus modifies cellular processes before hostresistance responses are activated. Collectively, our findings suggest aunique and powerful mechanism by which some viruses commandeer hosttranscription machinery through direct modifications of histones andthereby govern a wide range of chromatin-mediated cellular processes.

Example 2: Expression of vSET in a Host Cell Results in Global GeneSilencing that Triggers Cell Cycle Arrest or Apoptosis

Transgenic Arabidopsis plants were generated containing a beta-estradiolinducible vSET expression vector as well as another transferred pSGCOR1vector, which carries a promoter from the Arabidopsis ubiquitin genedriving the expression of a fusion protein that is made up of GAL4,estrogen receptor, and VP-16. The latter transferred DNA confershygromycin resistance to the plant. The transformed plants livenormally, exhibiting a wild-type phenotype prior to the induction ofvSET expression (FIG. 8a , control). About 10 days after beta-estradiol(10 μM) induced expression of vSET, the plants exhibited massive celldeath as shown in FIG. 8b -c.

Example 3: Transcriptional Silencing of Human Disease Causing Genes byvSET

The ability of vSET to repress Ga14-based promoter system and HOXA7promoter at the transcriptional level through its histone H3 lysine 27methylation (see, Example 1, supra; and Mujtaba et al., Nat Cell Biol.,10(9):1114-22 (2008)), prompted the examination of whether and how vSETcould silence genes that have been implicated in etiology andpathogenesis of human diseases such as androgen receptor (AR) inprostate cancer (Heinlein et al., Endocr Rev, 25(2):276-308 (2004)) aswell as selected genes in other diseases as listed in Table 1.

Methods

In this study, human HeLa cells were co-transfected with selected genepromoters (Table 1) in tandem with the luciferase gene with either anempty vector (pcDNA4TO) as a control or with vSET cloned into thepcDNA4TO vector. vSET expression was induced after treating the HeLacells with 5 μg of tetracycline. Target gene expression was thenassessed by measuring luciferase values using a dual luciferase assaykit (Promega, Cat# E1510), and normalized with the renilla luciferasevalue.

Results

As shown in FIG. 1, co-transfection of pcNDA4TO expression andluciferase reporter gene has little effects on target gene expression.However, upon expression induced tetracycline, vSET can effectivelyrepress transcription of AR (prostate cancer) (Heinlein et al., EndocrRev, 25(2):276-308 (2004)), HOXA7 (ovarian cancer) (Naora et al., ProcNatl Acad Sci USA, 98(26):15209-14 (2001)). HOXA9 (blood disorders)(Kroon et al., EMBO J, 20(3):350-61 (2001)), HOXC8 (prostate cancer)(Waltregny et al., Prostate, 50(3):162-9 (2002)), RAR (leukemia) (Zelentet al., Oncogene, 20(49):7186-203 (2001)), cyclin D (cancers) (Knudsenet al., Oncogene, 25(11):1620-8 (2006)), and NK-κB (inflammation)(Christman et al., Intensive Care Med, 24(11):1131-8 (1998)) See alsoTable 1. Moreover, vSET H3K27 methylation activity also resulted in anincreased transcription of E. cadherin (Sawada et al., Cancer Res,68(7):2329-39 (2008)) and M50/beta catenin (Lin et al., Proc Natl AcadSci USA, 97(8):4262-6 (2000)) (see FIG. 1), suggesting that these genesare suppressed by other regulatory genes, which transcription is likelytargeted by vSET. Taken together, this study demonstrates that vSET caneffectively suppress transcription of genes implicated in human diseasesthrough its activity of methylation of histone H3 lysine 27 at chromatinsites where the target genes are resided.

As such. vSET can be developed into a target gene specific silencingtechnology. This will be achieved by fusing vSET to a DNA bindingprotein domain that recognizes specifically the promoter sequence of agiven target gene, or to a histone binding protein domain that interactswith a core histone H3 or H4 carrying a distinct post-translationalamino acid modification at the target gene site. Furthermore, vSET willbe engineered to possess regulatory capacity via mutagenesis of aminoacid residues at the enzyme active site or methyl donor(S-adenosyl-methionine) co-factor binding site, in which a novelsmall-molecule chemical compound could be developed to control theenzymatic activity of vSET in a spatial and temporal manner. As such, avSET-based target specific gene silencing technology could be developedinto novel disease therapies for various human diseases by silencingdisease-causing gene expression.

TABLE 1 Genes Selected for vSET Transcriptional Silencing Analysis GeneHuman Disease References Androgen Prostate cancer Heinlein et al., End.Reviews. Receptor 2004, 25 (2): 276-308 HOXA2 Developmental Chatonnet etal., Neural disorder Development 2007, 2: 19 HOXA5 Breast cancer Ramanet al., Nature 405, 974-978 HOXA7 Ovarian cancer Naora et al., PNAS,2001, 18; 98: 15209-14 HOXC8 Prostate cancer Waltregny et al., Prostate,2002, 15; 50: 162-9. HOXA9 Blood disorders Kroon et al., The EMBO J.2001, 20; 350-361 Retinoic Leukemia Zelent et al., Oncogene, 2001, 20;Acid 49: 7186-03 receptor (RAR) Retinoic Cancers Altucci et al., NatureRev Drug Acid X Dis. 2007, 6; 793-810 receptor (RXR) Cyclin D CancersKnudsen et al., Oncogene, 2006, 25; 1620-1628 E Cadherin Breast andovarian Sawada et al., Cancer Research, cancers 2008, 68; 2329 NF-κBInflammation Christman et al., Inten. Care Med. 1998, 24: 1131-1138M50/Beta Cancers Lin et al., PNAS, 2000, 97: 4262- Catenin 4266

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

What is claimed is:
 1. A pharmaceutical composition for humanadministration comprising a purified fusion protein comprising aChlorella virus SET domain of a viral histone lysine methyltransferaseprotein (vSET), or of a vSET-like protein, fused to a protein that bindsto a target gene, in a pharmaceutically acceptable carrier.
 2. Thecomposition of claim 1, wherein the vSET protein comprises an amino acidsequence defined by SEQ ID NO:2.
 3. The composition of claim 1, whereinthe purified fusion protein further comprises a nuclear localizationsignal.
 4. The composition of claim 3, wherein the nuclear localizationsignal is a Lys-Arg-Met-Arg (KRMR) (SEQ ID NO:3) peptide.
 5. Thecomposition of claim 1, wherein the Chlorella virus SET domain of aviral histone lysine methyltransferase protein (vSET) or of a vSET-likeprotein is covalently linked to the protein that binds to a target gene.6. The composition of claim 1, wherein the target gene is a cytokine, anoncogenic gene, a homeodomain gene, a transcription factor, a receptor,a regulatory gene, a cell cycle regulating protein or a viralreplication gene.
 7. The composition of claim 6, wherein the target geneis a cytokine selected from the group consisting of TNF-α, TGF-β, IFN-γ,IL-2, IL-10 and any combination thereof.
 8. The composition of claim 6,wherein the target gene is an oncogenic gene selected from the groupconsisting of MDM2, Src tyrosine kinases, Ras kinases, receptor tyrosinekinases, epidermal growth factor receptors (EGFRs), platelet-derivedgrowth factor receptors (PDGFRs), vascular endothelial growth factorreceptors (VEGFRs) and any combination thereof.
 9. The composition ofclaim 6, wherein the target gene is a homeodomain gene selected from thegroup consisting of HOXA2, HOXA5, HOXA7, HOXA9, HOXB9, HOXC6, HOXC8,HOXD8, Hey1 and any combination thereof.
 10. The composition of claim 6,wherein the target gene is a transcription factor selected from thegroup consisting of myc, NF-κB and combination thereof.
 11. Thecomposition of claim 6, wherein the target gene is a receptor selectedfrom the group consisting of an androgen receptor, a retinoic acidreceptor (RAR), a retinoic acid x receptor (RXR) and any combinationthereof.
 12. The composition of claim 6, wherein the target gene is aregulatory gene selected from the group consisting of E cadherin,M50/Beta-catenin and combination thereof.
 13. The composition of claim6, wherein the target gene is a cell cycle regulating protein selectedfrom the group consisting of any cyclin D proteins and any combinationthereof.
 14. The composition of claim 6, wherein the target gene is aHIV transcriptional activator protein tat.
 15. The composition of claim1, wherein the protein that binds to a target gene is selected from thegroup consisting of transcription factors, enhancer proteins, suppressorproteins, the DNA binding domains thereof and any combination thereof.16. The composition of claim 15, wherein the protein that binds to atarget gene is selected from the group consisting of NF-kB, HOXC6,HOXC8, the DNA binding domains thereof and any combination thereof. 17.A pharmaceutical composition for selectively inhibiting transcriptionalexpression of a target gene in a subject in need thereof, comprising: i)a purified fusion protein comprising: a) a Chlorella virus SET domain ofa viral histone lysine methyltransferase protein (vSET) or of avSET-like protein, covalently linked to b) a protein that binds to thetarget gene, and ii) a pharmaceutically acceptable carrier.